JP2000505312A - Target nucleic acid sequence amplification - Google Patents

Abstract

  (57) [Summary] A method is provided for rapidly and substantially equilibratingly separating and amplifying sequence information of a target nucleic acid sequence located within a single-stranded or double-stranded polynucleotide. This method processes the double-stranded DNA so that the nicked site ends, nicks these sites, and extends from these nicked portions, thereby displacing any polynucleotide present. It is based on a series of steps. Also provided are methods for detecting a polynucleotide using the methods of the invention. Kits are also provided that combine components commonly used in practicing the present invention.

Description

DETAILED DESCRIPTION OF THE INVENTION                         Target nucleic acid sequence amplification                             Field of the invention   The present invention provides the use of only two amplification primers under substantially equilibrium conditions, Enable detection of the presence of a target nucleic acid sequence from a mixture of polynucleotides Exponential amplification of DNA or RNA. In addition, the present invention It also relates to a method for producing an amplification medium for a target nucleic acid used in a breadth process. The present invention relates to the identification of target nucleic acid sequences characteristic of organisms and genetic Even if it is present in trace amounts in a mixture of otides and polynucleotides, Is useful because it allows amplification and detection of Particularly useful of the method of the present invention Uses include pathogens that can cause disease in humans, animals or plants, e.g. For example, the presence of a target nucleic acid sequence characteristic of a virus, bacterium, parasite or fungus There are tests for food, biological fluids and tap water.                             Background of the Invention   Amplification, detection and quantification of specific target nucleic acid sequences can be attributed to microbial classification, inborn errors of metabolism Identification of genetic abnormalities, including infectious disease diagnosis, forensic analysis, environmental testing, and It is useful in various aspects, including research involving biobiology and cell biology.   The first approach to polynucleotide detection involves differentiation based on sequence specificity. I do. In general, polynucleotide assays rely on sequence specificity (ie, complementarity). To utilize the ability of the nucleic acid to hybridize or anneal. Yes The ability of a polynucleotide to be detected by hybridization depends on the sensitivity of the assay. Exist. The sensitivity in these hybridization assays is the signal Polynus used to search for complementary sequences in the sample along with the components used in the synthesis It is a function of the specific activity of the probe system containing nucleotides. However, poly The specific activity of the nucleotide probe system is limited. Hybridization To further enhance sensitivity in the assay, one of skill in the art will recognize the target nucleic acid sequence to be assayed. Began to increase.   To improve the detection of target nucleic acid by increasing the amount of target molecule at the beginning of the assay, Various amplification techniques have been devised. These techniques include strand displacement amplification ( I.e., SDA), polymerase chain reaction (i.e., PCR), Strand reaction (i.e., RT-PCR), nucleic acid sequence base amplification (i.e., NASBA), self-sustaining There are sequence replication (ie, 3SR), and ligase chain reaction (ie, LCR). This Each of these techniques produces large quantities of target polynucleotides, Increases sensitivity in nucleotide detection assays.   Strand displacement amplification (SDA) is described by Walker et al., Proc. Natl. Acad. Sci. (USA) 89: 392-39. 6 (1992), Walker et al., Nucl. Acids Res. 20 (7): 1691-1696 (1992), and U.S. Patents Nos. 5,270,184, 5,422,252, 5,455,166 and 5,470,723 I have. SDA is a DNA copy of a single-stranded or double-stranded target polynucleotide fragment Is an isothermal amplification technique that produces   Walker et al., Proc. Natl. Acad. Sci. (USA) 89: 392-396 (1992) and U.S. Pat. The first SDA methodology described in US Patent No. 5,166 describes a single-stranded or double-stranded target polynucleotide. Amplification of the entire sequence of a nucleotide fragment is disclosed. In this method, two primers ー Each primer has a target binding region at its 3 'end. Each plastic Positioned towards the 5 'end of the immer is the restriction that you can put a nick It is a single-stranded sequence corresponding to the endonuclease recognition site. These recognition sequences It need not be complementary to any sequence in the target polynucleotide fragment, and The 3 ′ end of the original double-stranded target polynucleotide fragment, or the original single-stranded target polynucleotide It is designed to engage the 3 'end of a nucleotide fragment and its complement. This In such a position, the target polynucleotide is engaged with the 3 'end of the primer. Unbound sequences are the free 3 'ends of both primers and each of the target polynucleotides. Serves as a template for enzymatic extension from each. These primers Must engage with any 3 'end of the oligonucleotide fragment (and its target complement) The nicked site (this is double-stranded) Must produce) any double stranded that is amplified in the SDA method Of the nucleotide sequence at both 3 'ends of the target polynucleotide fragment Required. For amplification of any single-stranded target polynucleotide, the 5 ' The nucleotide sequence at the 3 'end must be known. The sample you want to analyze Various collections, such as those found in biological fluids, tissues, food and tap water A mixture of polynucleotide fragments from various sources at various degradation or fragmentation stages. In such a case, it is difficult to know the sequence of the nucleotide terminus. Follow At the very end of the target polynucleotide fragment which is thought to contain the target nucleic acid sequence. For amplifying a target nucleic acid sequence without requiring knowledge of the nucleotide sequence of the target It is an object of the present invention to provide   Improvements to the SDA Act in U.S. Patents 5,270,184 and 5,422,252 Has been described. The '184 patent describes an amplification method involving four primers. ing. Amplification primers (S₁And S_Two) And '184 In the patented method, two bumper primers (B₁And B_Two) Is used. These four In designing the sequences of the two primers, the method of the '184 patent uses an internal target nucleic acid. At each end of the sequence, and furthermore, in the region adjacent to these terminal target sequences. Knowledge of the nucleotide sequence is required. Such need is for the diagnosis of pathogenic viruses Target genomic parts, such as parts or alleles associated with human disease In view of the time and effort required to fully identify the nucleotide sequence, It is an obstacle in advancing the breadth. In addition, the method of the '184 patent requires Incorrect annealing due to some of the four primers in the primer set Frequent, unnatural, and unexpected results have occurred.   Other improvements to the original SDA method are described in the '252 and' 723 patents, "Multiplex" amplification. The methods described in these patents are subject to one or more 6 primers (2 initial SDA primers) Imer, two bumper primers, and two adapter primers) Features beyond those required by the original SDA method or the method described in the '184 patent, That is, polynucleotides of the target genome for constructing these six primers Need more appropriate features as sources. In addition, countless plastics in the reaction mixture Immers will increase the likelihood of antagonizing the amplification reaction. Accordingly, the present invention The target should be sufficient to allow binding of the two primers under standard conditions in the art. Limits the required features of the target genome for the terminal sequence of the target nucleic acid sequence It is in.   Polymerase chain reaction (PCR) amplifies target sequences found in DNA molecules. To provide an alternative. Saiki et al., Science 239: 487-491 (1989), U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159. In the PCR method, two Although only primers are used, the target of amplification by the PCR method is a specific region of the target DNA. In addition, in actual use, a heat cycle device is required. Therefore, An object of the present invention is not only to amplify both target DNA and RNA, but also to It is an object of the present invention to provide a method that does not require a cooling device or a heat cycle device.   RT-PCR is a modified version of PCR that involves a target sequence found in an RNA molecule. To amplify. Myers et al., Biochemistry 30: 7661-7666 (1991) and U.S. Pat. No. 5,310,652 and No. 5,407,800. In RT-PCR, reverse transcription of target RNA As the cycle begins, a cDNA product is generated that is amplified by PCR. Same as PCR Similarly, in RT-PCR, a thermocycling device can be used to automatically apply the necessary temperature change. This is the first time it can be put to practical use. Therefore, the object of the present invention is to Provided is a method for amplifying a target polynucleotide that does not require a cycle device. It is in.   Other improved amplification methods have other disadvantages. Nucleic acid sequence base amplification (NASBA) and self-sustaining sequence replication (3SR) amplify target RNA molecules and Mainly produce single-stranded and double-stranded DNA. NASBA is based on Kievits et al., J. .Virol. Methods 35: 273-286 (1991) and U.S. Patent Nos. 5,130,238 and 5,409,8. It is described in No. 18. 3SR is described in Guatelli et al., Proc. Natl. Acad. Sci. (US A) 87: 1874-1878 (1990). A similar method is described in U.S. Patent No. 5,399,491. No. These methods have several features, namely Reverse transcription of target RNA molecules, provided by properly designed primers Double-stranded synthesis to produce a double-stranded cDNA containing the selected promoter, and And the use of promoters to produce RNA transcripts I have. This method relies on mesophilic microbial enzymes that become unstable at elevated temperatures. This Instability limits the maximum allowable temperature of the NASBA method to about 41 ° C. But this At relatively low temperatures, hybridization of the primers Accuracy will be reduced. Therefore, in this method, the frequent Problems caused by introduction, i.e. The inconvenience of the motor being settled and the promoter being generated at an undesired position Occurs. In addition, this method can be used to eliminate imbalances in the reaction components, especially enzyme activity levels. Extremely sensitive. In addition, the main product is RNA, which is less stable than DNA Ie, a polynucleotide.   Another method for amplifying nucleic acid sequences is the ligase chain reaction (LCR), First, it was proposed by Wu et al., Genomics 4: 560-569 (1989), The method of Ip was introduced by Barany, PCR Methods and Applications 1: 5-16 (1991). Was through. The LCR method utilizes two pairs of primers. In the LCR method, a pair of The primer is annealed to a proximate location of the target nucleic acid sequence. Other pair of primers Is located close to the complement of the target nucleic acid sequence region to which the first primer is attached. Annealing. Targets in the LCR method are exponentially amplified, but target-dependent Due to the nature of ligation, this method lacks certainty. further, Almost all of the amplified product is composed of pre-existing primer sequences. You. In addition, as with PCR, LCR uses thermal cycling and thermal Requires cycle equipment. Therefore, the purpose of the present invention is to provide a thermal cycle and four Primers that not only make the immersion unnecessary, but also contain a large number of amplified products -To provide a method that is not occupied by sequences.   Therefore, it is versatile, reliable and simple in the art. There is a need for a method of amplifying a nucleic acid sequence. Such a method involves both DNA and RNA. Using the minimum number of primers under substantially equilibrium conditions. And should be exponentially amplified.Summary of the Invention   The present invention relates to a method of substantially equilibrating under substantially equilibrium conditions consisting of a substantially constant temperature and reagent concentration. Amplification of nucleic acid sequences using enzyme-catalyzed extensions obtained from two amplification primers The above-mentioned needs are satisfied by providing methods and kits. Book A first aspect of the invention provides for the use of a semi-modified between two restriction endonuclease recognition sites. Isolating a copy of a target nucleic acid sequence from a substantially large polynucleotide in This method is applicable to both RNA and DNA, and single-stranded and And double-stranded targets. If the target nucleic acid sequence is considered a single-stranded nucleic acid sequence If both the copy of the target nucleic acid sequence and its complement Between the restriction endonuclease recognition sites. Single-stranded polynucleotides are prepared by standard techniques in the art. Obtained from oligonucleotides or double-stranded polynucleotides. Second aspect of the present invention Likewise define the opposite end of the target nucleic acid sequence, if present, and preferably With exponential amplification of isolated copies relatively independent of the predominant side reaction Two A method for amplifying a separated copy of a target nucleic acid sequence in the presence of a primer. Furthermore, the first and second aspects can be performed individually or simultaneously in the same reaction mixture. Application is also included in the scope of the present invention.   Specifically, according to the first aspect, the target present in the single-stranded polynucleotide A method for separating copies of a nucleic acid sequence, wherein the separated copy comprises a target nucleic acid. Suitable for sequence amplification, the method comprises:           (a) First amplification at the 3 'end of a target nucleic acid sequence within a single-stranded polynucleotide The primers hybridize, i.e., the hybridization For forming a hybridized first amplification primer, wherein the first amplification The primer comprises an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end, The 3 'end of the first amplification primer is complementary to the 3' end of the target nucleic acid sequence, and The 5 'end of the amplification primer contains the first recognition sequence for the first restriction endonuclease. The endonuclease recognizes the double-stranded, semi-modified One that can nick one strand of the site;           (b) extending the hybridized first amplification primer from its 3 ′ end, That is, the single-stranded polynucleotide has complementarity and is bound thereto First double-stranded polynucleotide having a modified first polynucleotide extension product To form a deoxyribonucleotide using the target nucleic acid sequence as a template to form Do triphosphate, modified deoxyribonucleoside triphosphate, and deoxyribo In the presence of a first polymerizing enzyme capable of polymerizing nucleoside triphosphate, its 3 ′ Extending from the end the hybridized first amplification primer;           (c) from the modified first polynucleotide extension product, Separating the single-stranded polynucleotide;           (d) A second amplification plasmid is inserted at the internal site of the modified first polynucleotide extension product. Hybridizes the primers, i.e., the hybridization The second amplification primer is for forming a second amplified primer. The primer comprises an oligodeoxyribonucleotide with a 3 'end and a 5' end, The 3 'end of the two amplification primer is complementary to the 3' end of the complement of the target nucleic acid sequence, It can also hybridize to it, the 5 'end of the second amplification primer Has a second recognition sequence for a second restriction endonuclease; Clease nicks on one strand of the double-stranded, semi-modified recognition site that it recognizes. Can be inserted;           (e) elongating the hybridized second amplification primer, i.e. Modified second polynucleotide hybridized to the decorated first polynucleotide extension product To form a second double-stranded polynucleotide containing a nucleotide extension product, Oxyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate And the deoxyribonucleoside triphosphate in the presence of the polynucleotide template Including a mixture of secondary polymerases capable of polymerizing and displacing chains. Of the hybridized second amplification primer from its 3 'end in the reaction mixture Elongation, wherein the modified second polynucleotide extension product is a single-stranded polynucleotide. Is separated from the nucleotide and opposes between the second recognition sequence and the complement of the first recognition sequence. A modified first polynucleotide comprising a copy of the target nucleic acid sequence located at the extreme end Between the first recognition sequence in the extension product and the first recognition sequence in the second polynucleotide extension product The complement is a double-stranded, semi-modified recognition site for the first recognition endonuclease To form; and           (f) Double-stranded semi-modified recognition for primary recognition endonuclease Nick one strand of the site, i.e., the first restriction endonuclease, Xyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate, And a double strand for the first recognition endonuclease in the presence of the second polymerase Nicking one half of the semi-modified recognition site of The 3 'end generated by the insertion is extended, thereby modifying the downstream modification. Any portion of the first polynucleotide extension product can be replaced, and Nicks for each of the second and second restriction endonucleases A target nucleic acid sequence defined at opposite ends by first and second recognition sites Containing a separated copy of the target nucleic acid sequence hybridized to a separated copy of the complement of Forming three double-stranded polynucleotides.   Identify copies of an amplifiable target nucleic acid sequence found in a single-stranded polynucleotide. The above-described method for separation involves a single reaction mixture containing all of the reactants listed above. It can be implemented with things. In the latter preferred embodiment, the above-described hybridization and expansion Length and nick steps all require steps other than preparing the reaction mixture. Without spontaneous progress in the reaction mixture. However, in the manner described above Single-stranded nuclei for spontaneously proceeding hybridization, extension and nicking steps In order to obtain an acid substrate, the desired single-stranded polynucleotide was contained or The reaction mixture suspected of containing the polynucleotide comprises at least one separation step. In the same separation step, any double-stranded nucleic acids in the reaction mixture Are also separated into single-stranded nucleic acids that can hybridize with a suitable target search primer, The primer then extends in the 3 'direction across the target template.   Thus, Applicants' preferred embodiment is found within a single-stranded polynucleotide. A method for separating a copy of a target nucleic acid sequence, wherein the separated copy comprises a target nucleic acid sequence. Suitable for use for sequence amplification, the method comprises:           (a) preparing a reaction mixture, the reaction mixture comprising:           (i) The target nucleic acid sequence is contained at a position other than the terminal in the single-stranded polynucleotide. Or a sample suspected of containing the target nucleic acid sequence;           (ii) Hybridization using a single strand of the polynucleotide as a template The deoxyribonucleoside triphosphate by extension of the primer The first polymerase that can be combined and the hybridized primer Deoxyribonucleoside triphosphate can be polymerized by lengthening, And a second polymerase that can also displace the strand if the target nucleic acid is DNA, The first polymerase can optionally be the same as the second polymerase. Synthase and second polymerase;         (iii) Deoxyribonucleoside containing modified deoxyribonucleoside triphosphate A mixture of nucleoside triphosphates;           (iv) hybridizable to a target nucleic acid sequence and hybridized thereto A first amplification primer capable of forming a primer having a 3 ′ end and a 5 ′ An oligodeoxyribonucleotide with an end, 3 'of the first amplification primer The end is complementary to the 3 ′ end of the target nucleic acid sequence, and the 5 ′ end of the first amplification primer is One strand of a double-stranded semi-modified recognition site for the first restriction endonuclease Recognition sequence for the first restriction endonuclease that can nick A first amplification primer having an array; and           (v) a second containing an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end An amplification primer, wherein the 3 'end of the second amplification primer is complementary to the target nucleic acid sequence. Complementary to and hybridizable to the 3 'end of the body The 5 'end of the second amplification primer is a second recognition for a second restriction endonuclease. A double-stranded semi-modified that the endonuclease recognizes Restriction endonuclease that can nick one strand of the identified recognition site A second amplification primer that is           (b) The means for separating double-stranded nucleic acids in the reaction mixture into single-stranded A reaction mixture; and           (c) Hybridization, primer extension, and nicking The reaction mixture is allowed to stand for a time sufficient to perform, thereby separating the target nucleic acid sequence. A double-stranded plasmid suitable for amplification, containing a separate copy of the copy and the complement of the target nucleic acid sequence. A polynucleotide extension product is generated, the double-stranded polynucleotide extension product comprising: At the opposite end, two for each of the first and second restriction endonucleases Defined by the first restriction site and the second restriction site into which the lock is inserted. .   The above-described method is hereinafter referred to as “short separation method, steps (a)-(c)”.   As a second aspect, the present invention relates to a target nucleic acid sequence obtained as described above. The separated copy is used as an intermediate in the polynucleotide reaction mechanism, that is, an amplification template. The present invention relates to a method for amplifying a target polynucleotide to be used. Therefore, the second aspect of the present invention Embodiments provide a method for amplifying a target nucleic acid sequence in a mixture of polynucleotides. In addition, the method comprises the steps (a)-(f) above;           (g) increasing the number of separate copies of the target nucleic acid sequence or its complement Width, ie, having DNA polymerase activity, and capable of displacing the strand. Enzymes, deoxyribonucleoside triphosphates and modified deoxyribonucleotides A third double-stranded polynucleotide in the presence of a mixture of osidotriphosphates; Contacting the restriction endonuclease with the second restriction endonuclease, Amplify an isolated copy of the target nucleic acid sequence or its complement, thereby providing a first and The first and second recognition sequences of the first and second recognition sites are nicked, and the nick is inserted. The 3 'ends of these sites are extended to produce a new target nucleic acid sequence or its complement. And the downstream copy of the target nucleic acid sequence or its complement Substituted; and           (h) when sufficient to produce a detectable amount of a separated copy of the target nucleic acid sequence Continuing the step (g) for a while.   Alternatively, a method of amplifying a target nucleic acid sequence located within a polynucleotide sequence is The above steps (g) and (h) are referred to as steps (d) and (e), respectively, as a short separation method, steps (a) to (c). Add and replace the execution procedure in each step (g) and (h) in one place Will be implemented. First, in step (g), `` Connect the third double-stranded polynucleotide The expression `` touch '' refers to `` contacting the double-stranded polynucleotide extension product '' in step (d). To the notation. Next, the notation of "step (g)" in step (h) is referred to as step (e). ) In step (d). The amplification method described in this paragraph, It is referred to as “short amplification method, steps (a)-(e)”.   In a third aspect of the invention, the presence of the amplified copy of the separated target nucleic acid sequence is Separated by using conventional DNA detection and identification methods, For example, detection of species-specific genotypes characterized by target nucleic acid sequences has been The gene type Identify or indicate that a particular organism has been included or contacted the tested sample. Hinting. Thus, the presence of a detectable amount of a separated copy of the target nucleic acid sequence, e.g., The method of determining the presence or absence of association between the material and a specific genotype or organism is described in steps (a) to (h) above. And the following step (i):           (i) determining the presence of a detectable amount of the separated copy of the target nucleic acid sequence and detecting If the presence of a possible amount of separated copies of the target nucleic acid sequence is observed, Include additional steps to indicate that linked genotypes or organisms are present in the sample No.   In another embodiment, the method for determining the presence of a target nucleic acid sequence comprises the step (i) ) As step (f), a short amplification method, and a method having a configuration added to steps (a) to (e). detection The latter method for determining the presence of a possible amount of a target nucleic acid sequence is hereinafter referred to as the “short determination method, (A)-(f) ".   In a preferred embodiment, an amplicon showing complementarity to each primer Of the polynucleotide portion (ie, the copied or amplified strand) Synthesized using deoxyribonucleoside triphosphate modified at the nucleotide moiety It is qualified because it was done. As a result, the restriction endonucleus located at the end is The enzyme recognition site is substantially semi-modified, and the region of the terminal recognition site is Substantially unmodified nucleotide components assisted by the complement and the modified complement including. As substantially semi-modified sites, these restriction sites, for example, Make sure that the nucleotides immediately 3 'to the cleavage site on one strand are modified. Thus, a nick can be made (that is, a single strand is easily cleaved).   Restriction endonuclease recognition sites at each end of the double-stranded polynucleotide are different. Design for restriction endonucleases You can also. Only one restriction endonuclease recognition site can be nicked Nicks can be nicked with the appropriate restriction endonucleases described above. further, If one or more nicking enzymes are used, at some point during the amplification procedure For example, by heating or chelation of an essential metal cofactor, One of these enzymes can be selectively inactivated. Alternatively, use different enzymes A small amount can be added to the reaction system. This way, in this method, one chain This results in asymmetric amplification of Has polymerase activity and displaces strand And preferably lacks effective exonuclease activity Other enzymes nick each of the terminal restriction endonuclease recognition sites The internal 3 'end generated by this is extended. Polynucleus extending from the nick Otide synthesis is a polynucleotide that terminates at the internal 5 'end created by nicking. Displaces the tide chain. The sequence information of the separated target nucleic acid sequence is It is amplified by repeating the conversion process.   A third aspect of the invention relates to amplification of target DNA or RNA involved in certain conditions. And the detection of the presence of a target polynucleotide involved in a condition (including but not limited to Use to determine the existence of such conditions based on considerations that include, but not limited to) It is in.   In a preferred embodiment, the method of the invention is carried out in environmental water based on the isolation and amplification of target nucleic acid sequences (if any) characteristic of Cryptosporidium parvum. Used to determine the presence of Cryptosporidium parvum. In the method of the present invention In another preferred embodiment, the degree of progression of the disease state is assessed. For example, a book The method of the invention is used to detect specific alleles associated with human breast cancer. Can be other In an embodiment, the method of the present invention comprises a Salmonella spp., A Clostridium spp. For example, Clostridium botulinum or Clostridium perfrigens), processed foods, meat, and other bacterial species lurking in dairy products. Can be used to quickly detect the location. In addition to these applications, The method detects nucleic acids of viruses that cause disease in humans, other animals, and plants. Can also be used to get out.   Another aspect of the present invention relates to a kit comprising components for performing the method of the present invention. And this kit:           (a) Dehydration to produce an essential first product from the complement of the template A polymerizing enzyme capable of polymerizing xyribonucleoside triphosphates, A polymerizable enzyme that can be           (b) a pair of amplification primers including a first amplification primer and a second amplification primer Wherein the first amplification primer is other than the end of the large polynucleotide. A 3 ′ end that is complementary to the 3 ′ end of the target nucleic acid sequence located at A first restriction endonuclease that can nick the first recognition site Has a 5 'end with a first restriction sequence for the second amplification primer, A 3 ′ end that is complementary to the 3 ′ end of the complement of the target nucleic acid sequence and a semi-modified second recognition Second for restriction endonuclease that can nick the recognition site A pair of amplification primers having a 5 'end with a restriction sequence;           (c) Nicking a substantially semi-modified restriction endonuclease recognition site One or two restriction endonucleases that can be           (d) deoxyribonucleoside triphosphate; and           (e) a modified deoxyribonucleoside triphosphate, Including.   As will occur to those skilled in the art, without departing from the spirit of the invention, for example, Other components, such as an opponent, and / or include, for example, ribonuclease H activity. Means for separating polynucleotide strands, such as means for separating polynucleotides, can also be added to the kit.   A unique advantage of practicing the present invention is that target nucleic acid sequences can be used in large quantities of single-stranded polynucleotides. Even if it is in the nucleotide, And / or any target nucleic acid in a mixture of polynucleotide fragments. The point is that the sequence can also be amplified. This amplification performance, which has a wide range of applications, To the entire sequence or subsequence of a given polynucleotide. Wide range of application Expands the horizons when planning an amplification plan, and provides a full-length target for amplification. And virtually eliminates the time and cost previously required to produce the target polynucleotide.   Another advantage of the present invention is that polynucleotide sequences can be amplified under substantially equilibrium conditions. There is to be able to. This substantially equilibrium condition is defined as substantially constant temperature and substantially constant temperature. Contains unmodified reaction components. The first step of the method of the invention involves the target nucleic acid sequence. Or a double-stranded polynucleotide fragment thought to contain the target nucleic acid sequence Or a single-stranded target polynucleotide hybridized to the first polynucleotide extension product Involves temperature changes due to denaturation of nucleotides, but subsequent amplification cycles Become unrelated to transformation. Further, in one embodiment of the present invention, the target polynucleotide If the strand is single-stranded, for example, RNA, there is no need to adjust the reaction temperature. It becomes even smaller. However, at any point after the reaction has proceeded, Equilibrium conditions are maintained because there is no need to adjust the dose. In addition, the reaction volume changes. The reagent concentration is adjusted by direct or indirect operator operation. Not. Therefore, the method of the present invention requires the cost of monitoring and detailed operation. Can also work perfectly.   Yet another advantage of the method of the present invention is that any primer can be used with two primers. It is also capable of amplifying a nucleotide sequence. In the present invention, an adapter, a bumper, Alternatively, use these primers in the intermediate amplification product without using ligation primers. Incorporates an array. Primers are usually prepared by costly synthetic routes. Since the primers required for amplification in the present invention can be minimized, The cost required to implement the invention will be reduced.   The myriad aspects and advantages of the present invention will become apparent with reference to the following drawings and detailed description. Will be.                         BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 outlines the method of the present invention.   FIG. 2 shows three reverse transcripts, AMV-RT (lanes 1-4) and MMLV-RT (lanes 5-8). From the probe extension assay using the Superscript II and Superscript II (lanes 9-12) 1 shows a radioactivity photograph of the filtration product.   FIG. 3 shows the radioactivity of the gel filtration product from the probe extension assay (lanes 1-9). The picture is shown.   Figure 4 shows a probe extension assay that measures amplification products in relation to target copy number. 5 is a radioactivity photograph of a gel filtration product from Astellas.                         Detailed description of the invention   The invention has various aspects. In one embodiment, the invention A single-stranded polynucleotide using conditions that are substantially equilibrium with the two amplification primers. The present invention relates to a method for separating a target nucleic acid sequence from an otide, comprising the steps of: Semi-modified to quickly obtain a copy of the amplifiable target nucleic acid sequence substantially free of The copy (and its complement) between the two restriction endonuclease recognition sites Are separated. The single-stranded polynucleotide containing the target sequence is a single-stranded polynucleotide. Otides, or double-stranded polynucleotide fragments or mixtures thereof, It can be derived by techniques well known in the art.   Specifically, in the first aspect, the present invention provides a method for preparing a single-stranded polynucleotide. A method for isolating a copy of the target nucleic acid sequence located at Is suitable for use for amplification of a target nucleic acid sequence, the method comprising:           (a) First amplification at the 3 'end of a target nucleic acid sequence within a single-stranded polynucleotide The primers hybridize, i.e., the hybridization For forming a hybridized first amplification primer, wherein the first amplification The primer comprises an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end, The 3 'end of the first amplification primer is complementary to the 3' end of the target nucleic acid sequence, and The 5 'end of the amplification primer contains the first recognition sequence for the first restriction endonuclease. The endonuclease recognizes the double-stranded, semi-modified One that can nick one strand of the site;           (b) extending the hybridized first amplification primer from its 3 ′ end That is, the single-stranded polynucleotide has complementarity and is bound thereto. First double-stranded polynucleotide having a modified first polynucleotide extension product The target nucleic acid sequence is used as a template to form a deoxyribonucleic acid Nucleoside triphosphate, modified deoxyribonucleoside triphosphate, and deo In the presence of a primary polymerizing enzyme capable of polymerizing xyribonucleoside triphosphate Extending the hybridized first amplification primer from its 3 'end;           (c) converting the single-stranded polynucleotide from the modified first polynucleotide extension product Isolating otide;           (d) A second amplification plasmid is inserted at the internal site of the modified first polynucleotide extension product. Hybridizes the primers, i.e., the hybridization The second amplification primer is for forming a second amplified primer. The primer comprises an oligodeoxyribonucleotide with a 3 'end and a 5' end, The 3 'end of the two amplification primer is complementary to the 3' end of the complement of the target nucleic acid sequence, It can also hybridize to it, the 5 'end of the second amplification primer Has a second recognition sequence for a second restriction endonuclease; Clease nicks on one strand of the double-stranded, semi-modified recognition site that it recognizes. Can be inserted;           (e) elongating the hybridized second amplification primer, i.e. Modified second polynucleotide hybridized to the decorated first polynucleotide extension product To form a second double-stranded polynucleotide containing a nucleotide extension product, Oxyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate And the deoxyribonucleoside triphosphate in the presence of the polynucleotide template Including a mixture of secondary polymerases capable of polymerizing and displacing chains. Of the hybridized second amplification primer from its 3 'end in the reaction mixture Elongation, wherein the modified second polynucleotide extension product is a single-stranded polynucleotide. And separated from the second recognition sequence. Contains a copy of the target nucleic acid sequence located at the end opposite to the complement of the recognition sequence. The first recognition sequence and the second polynucleotide in the modified first polynucleotide extension product. The complement of the first recognition sequence in the otide extension product is the first recognition endonuclease. Forming a double-stranded, semi-modified recognition site for;           (f) Double-stranded semi-modified recognition for primary recognition endonuclease Nick one strand of the site, i.e., the first restriction endonuclease, Xyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate, And a double strand for the first recognition endonuclease in the presence of the second polymerase Nicking one half of the semi-modified recognition site of The 3 'end generated by the insertion is extended, thereby modifying the downstream modification. Any portion of the first polynucleotide extension product can be replaced, and Nicks for each of the second and second restriction endonucleases A target nucleic acid sequence defined at opposite ends by first and second recognition sites Containing a separated copy of the target nucleic acid sequence hybridized to a separated copy of the complement of Forming three double-stranded polynucleotides.   As used herein, the term “target nucleic acid sequence” or “target sequence” refers to the target sequence. It is known that large polynucleotides extend beyond both the 5 'and 3' ends. Refers to a nucleic acid sequence of interest that is or is considered to be there. In the present invention For a normal polynucleotide intended for use, the polynucleotide is Contains one to thousands more bases further from one or both of the 5 'and 3' ends of the target And, more generally, more than 10 to thousands of additional bases, and sometimes the target nucleic acid Includes more than 1000 bases further from both the 5 'and 3' ends of the sequence. In the field If present, the size of the polynucleotide containing the target sequence is Size and the amount of polynucleotide (ie, DNA or RNA) contained in the sample It will be appreciated that it depends on the degree of solution or fragmentation. Therefore, it is not used here. The term "polynucleotide" as used also includes polynucleotide fragments. Departure Target nucleic acid sequences suitable for use in the light include single-stranded RNA, double-stranded RNA, single-stranded D NA, double-stranded DNA, and DNA-RNA duplex. However, To perform the hybridization of the first and second amplification primers, Any double-stranded polynucleotide must be converted to its corresponding single-stranded form. No. Polynucleotides containing a target nucleic acid sequence are usually Is present in a sample that contains a plurality of other polynucleotides that do not. target The nucleic acid sequence is usually a specific organism, animal, plant, bacterium, fungus, parasite or virus. This is an array characteristic of Luss. For example, they can distinguish the same species from each other, Or, for example, to distinguish one strain of the same bacterium from another, or, for example, Target nucleic acid sequence may be selected to identify genetic variants due to mutation . Suitable target nucleic acid sequences are found in biological fluids, tissues, food and tap water, etc. To pathogenic organisms such as pathogenic bacteria, viruses, molds, parasites and fungi Related or characteristic sequences.   Pathogenic cells that provide a target nucleic acid sequence suitable for amplification and detection by the method of the invention Examples of bacteria include Bacillus anthracis (Bacillus anthracis), Bacillus botulinus (Bacillus botulinas) ) (Clostridium botulinum (clostridium botulinum)), Bacillus dysenterea ), Bacillus enteritidis (Bacillus enteritidis) (Salmonella enteritidis (Salmonella enteritidis)), Bacillus pneumoniae (Bacillus pneumoniae) (Diplococcus pneumoniae) ae (Klebsiella pneumoniae)), Bacillus tetani (Bacillastetani) (Clostridium tetani (Clostridium tetani)), Bacillus typhosus (Bacillus typhos) (Salmonella typhosa (Salmonella typhosa)), Chlamydia oculogenitalis (Clamydia oculogenitalis), Clostridium perfringens (Clostridium perfringens), Hemophilus influenzae (Hemophilus influenza), Hemophilus pertus sis (Hemophilus spatsis), Mycobacteria tuberculosis (Mycobacterium tuberculosis), Neisse ria gonorrhoeae (Pseudomonas aeruginosa), S higella paradysenteriae (Shigella paradysenteriae), Staphylococcus aureus (Staphylococcus aureus) ), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus faecal is (Streptococcus faecalis), Streptococcus hemolyticus (Streptococcus hemolyticus), Stre ptococcus mitis (Streptococcus mytis), Streptococcus pyogenes (Streptococcus pyogenes) , Vibrio cholerae, Vibrio comma, Vibrio fetus ), Vibrio jejuni (Vibrio jejuni), Vibrio metchnikovii (Vibrio metnicophy), and There is Vibrio niger. Nucleic acid sequences characteristic of each of these bacterial species are well known in the art, All or part thereof can be used as a target nucleic acid sequence in the method of the present invention. example For example, the entire genome sequence of Hemophilus influenzae is referred to herein in its entirety. And reported in Fleischmann et al., Science 269: 496-512 (1995). Have been.   Can provide a target nucleic acid sequence for amplification and detection by the methods of the invention. Examples of viruses including retroviruses include HSV-I, HSV-II, HIV, CMV, There are measles, polio, hepatitis B, hepatitis C, and influenza. These will Each of Various characteristic nucleic acid sequences are well known in the art, Can be used as the target nucleic acid sequence in the method of the invention. For example, the full text Molecular Methods for Virus Detection (Dann y L. Wiedbrauk and Daniel H. (Fakas ed., AACC Press, Washington D.C., 1995) checking ...   The method of the present invention separates the target nucleic acid sequence or target sequence, Use two amplification primers under substantially equilibrium conditions where the components are substantially constant. And enzymatically amplify (ie, copy) the sequence information contained in these targets . Each of the two amplification primers has a polynucleotide bond located at the 3 'end. Region and restriction endonucleus that can contain a nick located 5 'end It contains a single strand of a protease recognition site (ie, a recognition sequence). Single-stranded polynucle The target nucleic acid sequence in the leotide serves as a template for DNA synthesis using primers. To be used. Next, the two chains of the new duplex are separated and each chain is cast independently. Each strand to one of the two primers in the method of the invention. Neil. Alternatively, a single-stranded polynucleotide containing the target polynucleotide And its remaining complement is used to synthesize a DNA copy of the target nucleic acid sequence. Used as a template for Restrictions that can put a nick at the end To generate double-stranded DNA containing a nuclease recognition site, only a few All you need is a kuru.   Nickable first and second restriction endonuclease recognizers The positions may be the same or different. In addition, unmodified primers The presence of at least one modified deoxyribonucleoside triphosphate In the presence By performing the primer extension reaction, these sites can be nicked. It is a part that can be done. Alternatively, the art of processing into modified chains that cannot be cleaved Provide the duplex polynucleotides in a chain specificity modification method using techniques well known in the art. can do.   Double stranded dissociation of target nucleic acid sequence adjacent to nickable site Once a DNA copy is generated, it is amplified under conditions where temperature and reagent components are substantially equilibrium. Progresses. First, a nick is added to each end of the double-stranded polynucleotide, and the nick The free 3 'end generated by the enzyme is extended by a polymerase capable of displacing the chain. Lengthened. Preferably, the polymerase is used to disrupt pre-existing polynucleotide chains. Rather, it is the DNA polymerase to be replaced. The displaced strand is used for other complementary amplification Anneals the primers and provides the opportunity to geometrically increase the amplification rate. The nicking, extension and replacement steps are, for example, a single step that does not require repeated manipulation interventions. This is repeated using the above reaction vessel.   Is known to have or is likely to have the target nucleic acid sequence The sample containing the polynucleotide is the target organism or the inhabited organism. Obtained from an environment known or believed to be inhabited by the organism. Can be Human or human samples containing or suspected of containing the target nucleic acid sequence Typical sources in animals or animals are blood, saliva, tears, cerebrospinal fluid, exudates, exudates, urine Biological fluids such as ascites, sputum, mucus, semen, bone marrow, feces; or lungs, liver , Kidney, spleen, heart, hair, skin and other tissues. Other sources of target nucleic acid sequences Is used for plants, soil, food (especially meat, cooked food, shellfish and dairy products) and Water.   In a preferred embodiment, the target nucleic acid sequence is Cryptospor Contains the nucleic acid sequence of idium parvum. 18S rRNA gene of Cryptosporidium parvum Is more preferred. Cryptosporidium parvum 18S rR The nucleotide sequence of the NA gene has 1750 nucleotides and It can be obtained from the base under the accession number of L16997. Cryptosporidium parv The sequence of the um 18S rRNA gene is set forth in SEQ ID NO: 1. Cryptosporidium p Samples for performing analysis on arvum usually include drinking water, surface water, and feces. There are stools.   As used herein, the term "amplification primer" refers to the term Oligo video for amplifying the target nucleic acid sequence located by primer extension A xyribonucleotide primer is meant. The 3 'end of the amplification primer is Hybridizes to the 3 'end of either the acid sequence or its complement. Amplification primer If the 3 'end of the primer is hybridized to the polynucleotide chain, The 5'-end of the immer, for example, hybridizes to the polynucleotide strand found in the sample. No need to soy. In addition, the 5 'end of the amplification primer should be minimal, Has recognition sequences for nucleases or restriction enzymes. This recognition sequence If the recognition site is a semi-modified site, "nicking" one strand of the DNA duplex For endonuclease. As used herein, the term "nick" Only one of the two strands of the semi-modified recognition site of the DNA duplex is restricted to the restriction endonucleus. Means to be "nicked" by the lase.   In the present invention, the first and second recognition sequences each for one or more restriction enzymes Having two primers, a first amplification primer and a second amplification primer. I'm using The first and second recognition sequences are the same or different Recognized And the first and second recognition sequences are identical or nickable. Differences are also included in the present invention. In addition, amplification primers Also having one or more recognition sequences for a restriction endonuclease recognition site, Included in the present invention. For example, a restriction endonuclease for nicking Other recognition sequences located 5 'to the recognition sequence for nicking A polynucleotide comprising an isolated copy of a double-stranded target nucleic acid sequence flanked by restriction sites The fragments are used for cloning.   Nick the primer binding region or target binding region of the primer. A single strand of sequence information corresponding to the restriction endonuclease recognition site In view of the potential non-hybridizing portion of each primer included, the present invention Under the conditions (see below) used to perform the primer Must be sufficient to ensure kneeling. Preferably, the The target binding portion of the primer has at least about 16 nucleotides and Target binding sites are responsible for at least about 40% of the primer nucleotides.   Preferred amplification primers are oligodeoxy Ribonucleotides, and in a double-stranded molecule, the primer is a double-stranded polynucleotide. Nick sites (ie, Nicked endonuclease recognition or restriction site) Inhibits restriction endonuclease cleavage. Therefore, properly formed double-stranded Enzymes with nick activity when primers are involved in oligonucleotides Has the effect of not inhibiting the cleavage of the primer polynucleotide at the nick site If it is a modified nucleotide, this modified nucleotide Included in preferred primers of the invention.   Can contain nicks recognized by different restriction endonucleases If a different site is introduced, preferably a different restriction endonuclease, e.g. For example, it can be selectively inactivated by heating or metal chelation. . According to an example, one primer is specific for the strand of the recognition site for BsiHKCI And the second primer is specific for a recognition site for AlwI. Al wI is denatured by incubation at 65 ° C for 20 minutes, whereas BsiHKCI is denatured By exposing the amplification reaction to conditions at 65 ° C for 20 minutes, One can be selectively modified. And such a reaction is the 5 'end side Asymmetrically amplify the polynucleotide chain having the BsiHKCI sequence. is there Alternatively, different restriction endonucleases can be added at different levels of activity. Wear. Apart from these considerations, a nicked, preferred restriction end A nuclease recognition site is a non-palindromic site. With various restriction enzymes, U.S. Patent No. 5,270, each of which restriction sites are incorporated herein by reference. No. 184 (issued December 14, 1993) at column 10, lines 29-68.   The function of the first and second recognition sequences of the amplification primer is that of a semi-modified restriction endonuclease. It is to provide a template for generating a creatase recognition site. Used in this specification "Semi-modified restriction endonuclease recognition site" or "Semi-modified recognition A "sense site" is a double-stranded recognition sequence for a restriction enzyme, where one strand is usually At least one modified (or “triggered” molecule that prevents strand breakage by restriction enzymes Derived ") nucleotides. Modify the position where cleavage of the chain is blocked Nucleotides (also Is the other strand at the semi-modified recognition site that does not contain nucleotides) Puts "nick". Usually, a semi-modified recognition site is a single strand Involved in one or more hemi-phosphorothioated nucleotides Part. Appropriate restriction enzymes used to generate the modified recognition site The recognition sites and modified dNTPs are disclosed in U.S. Patents, the disclosure of which is specifically incorporated herein. No. 5,270,184 (issued December 14, 1993).   The function of deoxyribonucleoside triphosphate in the process of the present invention is To generate a complementary strand of the target nucleic acid sequence using the target, and use the target complement as a template. The role of the substrate for polymerizing an enzyme that produces a copy of the target nucleic acid sequence using What to do. A set of deoxyribonucleoside triphosphate monomers is preferred Preferably, at least one monomer is 2'-deoxyguanosine 5'-triphosphate, 2'-de Oxyadenosine 5'-triphosphate, 2'-deoxycytidine 5'-triphosphate, or thymidine A modified form of gin 5'-triphosphate. A preferred modified monomer is 2'-deo Xycytidine 5′-0- (1-thiotriphosphate) [ie, dCTP (αS)]. Other suitable In a specific embodiment, the modified monomer is 2′-deoxyguanosine 5′-0- (1- Thiotriphosphate), thymidine 5′-0- (1-thiotriphosphate), 2′-deoxycytidine 5′-0- (1 -Thiotriphosphate), 2'-deoxyuridine 5'-triphosphate, 5-methyldeoxycytidine 5'-triphosphate or 7-deaza-2'-deoxyguanosine 5'triphosphate. This It will be appreciated by those skilled in the art that the function of the modified monomer in the method of the present invention will work. If so, other modified monomers are included within the scope of the present invention. Of the modified monomer Function is the generation of a nickable restriction endonuclease recognition site Contributing to It is in. To provide this function, the modified monomer must be nicked Blocking the cleavage of one of two DNA strands containing a restriction endonuclease recognition site Must contribute. Usually, the modified nucleic acid monomer is Incorporated 3 'adjacent to the cleavage site of the nick enzyme at the It is not incorporated adjacent to the cleavage site at the 3 '. Modified nucleic acid monomer at this position Usually inhibits the cleavage of those chains. 3 'adjacent to the nick enzyme cleavage site The other chain lacking the modified monomer is cleaved and a nick is generated I do. Other modification procedures that prevent cleavage of one strand at the recognition site of the duplex have also been developed. Ming intends. Modifying one or more nucleic acid monomers is also within the scope of this invention. Is included. Further, those skilled in the art will appreciate that suitable nucleic acid monomers or monomers Can be used after synthesis to obtain a new mode of use of the modified nucleic acid monomer. Will come to mind.   In the present invention, deoxygenation is performed using an amplification primer and a target nucleic acid sequence as a template. Ribonucleoside triphosphate (and modified deoxyribonucleoside triphosphate (Acid) can be polymerized to the complementary strand of DNA. The polynucleotide synthesized in the first extension step is a polynucleotide sequence of the target nucleic acid. Becomes the complement of The polynucleotide synthesized in the second extension step is supplemented with the first extension product. And contains a copy of the target nucleic acid sequence. As a source of the first polymerase, There are prokaryotes, eukaryotes and viral organisms. The first polymerizing enzyme of the present invention Is produced recombinantly or synthetically, and is used for holoenzymes, enzyme subunits. Make up knits or their fragments. Primary polymerization enzymes are suitable for use in the present invention. Prior to the treatment, for example, a treatment with a reducing agent can be performed. The target nucleic acid is RNA In some cases, the first polymerase of the invention is preferably an RNA-dependent DNA polymerase. When the target nucleic acid is DNA, the DNA-dependent DNA polymerase preferable. Substantially lacks 5'-3 '(ie, 5'-> 3 ') exonuclease activity Also preferred are primary polymerization enzymes. Other preferred first polymerases are also substantially exonucleated. Lack of rease activity (3'-5 'and 5'-3' exonuclease activity). 45- Effective catalytic activity can be maintained even at a temperature of 85 ° C, more preferably 60 ° C A thermostable first polymerizing enzyme is more preferred. Use thermostable primary polymerase To synthesize polynucleotides at optimal temperatures for primer annealing Has the highest synthesis rate, which minimizes the production of unwanted polynucleotides. become. Preferred primary polymerases include avian myeloblastosis virus reverse transcriptase (AMV-R T), Moloney murine leukemia virus reverse transcriptase (MMLV-RT), Superscript I I (Superscript II) transcriptase (Life Technology, Rock, MD) Reverse transcriptase such as Bil) and E. coli DNA polymerase, E. coli. coli D NA polymerase (Klenow fragment), exo<sup>-</sup> E. coli DNA polymerase (cure) Nost fragment), Bst DNA polymerase, Taq DNA polymerase, Tfl DNA Remelase, and DNA polymerases such as Tth DNA polymerase But not limited to them. Reverse transcriptase activity and DNA polymerase activity The first polymerizing enzyme having both sides is also included in the scope of the present invention. According to such enzymes Reverse transcription activity is controlled by generating cDNA of the RNA target nucleic acid sequence. Copy acid sequence, while DNA polymerase copies DNA target nucleic acid .   In one step of the process of the present invention, the "modified first polynucleotide" Separate the target nucleic acid sequence from the otide extension product. " The target nucleic acid sequence is an RNA sequence When the modified first polynucleotide extension product is a cDNA, One means for separating a target nucleic acid sequence from a first polynucleotide extension product is an enzyme. Active. Preferably, the enzyme is partially or completely hydrolyzed to the target Digest the RNA strand containing the sequence. For example, RNA / DNA duplexes are ribonucleic Exposure to Hase H activity to produce single-stranded cDNA efficiently. In the art, Ribonuclease activity is determined by E. coli. unique enzymes such as E. coli ribonuclease H, Recognized as being brought about by its catalytically effective fragments . Ribonuclease H activity can be AMV-RT, MMLV-RT, or Versatile enzymes that express ribonuclease H activity, such as fragments thereof It is also brought. As an alternative to enzymatic separation for RNA, This can be achieved by physical means (eg, heating) or chemical means (eg, pH change). Separation step can be performed, but effectively separates the strands of the double-stranded polynucleotide Is less specific. The complementary strands of nucleic acids are separated from each other (ie, The use of enzymatic, physical and chemical means to Is well known.   In the second "extension" step of the process of the present invention, a second Two polymerases are used (i.e., complementary to the "template" and The strand that is replaced in the region where the complementary strand can hybridize to the template The "template" strand by primer extension to produce another strand that is identical Separation of hybridized polynucleotide chains). The second polymerase is the first extension product A polynucleotide that is complementary to, or the “template” described in parentheses above. Catalyzes the synthesis of When the target nucleic acid sequence is an RNA sequence, the activity of the second polymerase (ie, DNA Polymerase activity). As mentioned above, a single enzyme, reverse transcriptase It can have both activity and DNA polymerase activity. Examples of such enzymes Is well known in the art, for example, AMV-RT and MMLV-RT. Second growth A preferred second polymerase used in the long step is a DNA-dependent DNA polymerase. is there. Further, when the target nucleic acid is DNA, the first and second synthetases are the same. It is preferred to use the same enzyme. Examples of such polymerases include E. coli DNA Examples include a polymerase, a catalytic subunit or a fragment thereof. Good Preferably, this second polymerase has an exonuclease activity, in particular, 5'-3 'exo. It shall lack nuclease activity. For example, (Molecular Biology   Exo, commercially available from Resources Inc., Milwaukee, Wis.<sup>-</sup>  E. coli DNA polymerase (Klenow fragment) is a preferred second polymerase. You. The DNA polymerase used in one or both extension steps Inclusion of a polymerizing enzyme exhibiting heat resistance to temperature is also included in the scope of the present invention. Preferred High heat-resistant DNA polymerases include Tfl DNA polymerase and Taq DNA polymerase. Lase, Tth DNA polymerase and Bst DNA polymerase, It is not limited to these.   At the end of the nicking step, i.e. to separate copies of the target nucleic acid sequence At the end of step (f) in the human method, the Applicant's method specifies the target nucleic acid as the first strand Separate copy of sequence and complement of first strand hybridized to first strand as second strand Preparing double-stranded polynucleotides, including the first and second restriction ends No. that can put nicks for nucleases Each of the first and second recognition sites, at the opposite end, One chain is defined.   Isolates amplifiable copies of target nucleic acids found in single-stranded polynucleotides The above method for a single reaction mixture containing all of the reactants described herein Can be easily implemented. In the latter preferred embodiment, the above-described hybrid The size, elongation and nicking steps involve working steps other than preparing the reaction mixture. It proceeds spontaneously in the reaction mixture without need. However, as mentioned above For the first and second spontaneous hybridization steps in the process Applied to at least one separation step to prepare a suitable nucleic acid substrate In the separation step, any double-stranded nucleic acid in the reaction mixture is converted to a suitable target-search primer. The primers are separated into single-stranded nucleic acids that can hybridize with Extends in 3 'direction across the mold.   Accordingly, a preferred embodiment in Applicants' method for separating copies of a target nucleic acid sequence is described. When applied to actual processes, the first aspect of the applicant described above can be substantially shortened. You. Thus, Applicants' preferred embodiment is found within single-stranded polynucleotides. A method for isolating a copy of the target nucleic acid sequence, wherein the separated copy comprises Suitable for use for amplification of an acid sequence, the method comprises:           (a) preparing a reaction mixture, the reaction mixture comprising:           (i) The target nucleic acid sequence is contained at a position other than the terminal in the single-stranded polynucleotide. Or a sample suspected of containing the target nucleic acid sequence;           (ii) Hybridization using a single strand of the polynucleotide as a template Deoxy by extending the primer A first polymerizing enzyme capable of polymerizing siribonucleoside triphosphate; and Deoxyribonucleoside by extending the hybridized primer If the triphosphate can be polymerized and the target nucleic acid is DNA, the strand can be displaced. A second synthase that can also be used, wherein the first synthase is optionally combined with the second synthase. A first synthase and a second synthase, which can be identical;           (iii) Deoxy containing modified deoxyribonucleoside triphosphate A mixture of ribonucleoside triphosphates;           (iv) hybridizable to a target nucleic acid sequence and hybridized thereto A first amplification primer capable of forming a primer having a 3 ′ end and a 5 ′ An oligodeoxyribonucleotide with an end, 3 'of the first amplification primer The end is complementary to the 3 ′ end of the target nucleic acid sequence, and the 5 ′ end of the first amplification primer is One strand of a double-stranded semi-modified recognition site for the first restriction endonuclease Recognition sequence for the first restriction endonuclease that can nick A first amplification primer having an array; and           (v) a second containing an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end An amplification primer, wherein the 3 'end of the second amplification primer is complementary to the target nucleic acid sequence. Complementary to and hybridizable to the 3 'end of the body The 5 'end of the second amplification primer is a second recognition for a second restriction endonuclease. A double-stranded semi-modified that the endonuclease recognizes Restriction endonuclease that can nick one strand of the identified recognition site A second amplification primer that is           (b) for separating double-stranded nucleic acids in the reaction mixture into single-stranded Applying the reaction mixture to the means for:           (c) Hybridization, primer extension, and nicking The reaction mixture is allowed to stand for a time sufficient to perform, thereby separating the target nucleic acid sequence. A double-stranded plasmid suitable for amplification, containing a separate copy of the copy and the complement of the target nucleic acid sequence. A polynucleotide extension product is generated, the double-stranded polynucleotide extension product comprising: At the opposite end, two for each of the first and second restriction endonucleases Defined by the first restriction site and the second restriction site into which the lock is inserted. .   The above-described method is hereinafter referred to as “short separation method, steps (a)-(c)”. Of the above method In step (a), the reaction mixture is an aqueous or substantially aqueous medium suitable for nucleic acid polymerization. Placed inside. The use of such media is well-known in the art. Short The separation method, step (b) of steps (a) to (c), uses a means for separating double-stranded nucleic acids into single strands. The separated strands are used in large amounts of poly- Subsequent hybridization involved in separating copies of the target nucleic acid sequence from nucleotides It functions as a template for the lysis and extension reactions. Double-stranded nucleic acid Methods for separating into single strands are well known in the art, and An NA-RNA duplex is formed in step (a) and the target nucleic acid is a segment of RNA. Ribonuclease, for example, ribonuclease H, It can be included in or added to the compound. Ribonuclease for polymerizing enzymes It is within the scope of the present invention to provide a zebra-like activity. Separation detailed above Besides the means, there are chemical means such as pH and physical means such as heating. These technologies Are all well known in the art and will not be described further herein. Absent.   RN in which the target nucleic acid is recognized as an RNA duplex or an RNA-DNA duplex A or when the target nucleic acid is single-stranded DNA or RNA, Step (b) of “Separation method, steps (a)-(c)” is usually performed at a time. The target nucleic acid is D NA contained in the sample as a DNA duplex or an RNA-DNA duplex. In this case, the same step (b) is usually performed in two steps.   The amplifiable copies of the target nucleic acid sequence and its complement are Between the recognition sequence for the second restriction enzyme and the recognition sequence for the target nucleus. The isolated copy of the acid sequence, unlike the original target nucleic acid sequence, Isolated from the non-target nucleic acid sequence in it. Therefore, separation of the target nucleic acid sequence and its complement The copies can be amplified exponentially under substantially equilibrium conditions.   Thus, in the second aspect, the present invention provides a polynucleotide amplification reaction In the above, as an intermediate, that is, as an amplification template, obtained as described above The present invention relates to a method for amplifying a target nucleic acid sequence using a separated copy of the target nucleic acid sequence. Therefore The second aspect of the invention relates to amplifying a target nucleic acid sequence in a mixture of polynucleotides. The method comprises, in addition to steps (a)-(f) above:           (g) amplifying an isolated copy of the target nucleic acid sequence or its complement, i.e. , An enzyme having DNA polymerase activity and capable of strand displacement Siribonucleoside triphosphate and modified deoxyribonucleoside triphosphate A third double-stranded polynucleotide and a first restriction endonuclease in the presence of a mixture of The contact between the target nucleic acid sequence and the second restriction endonuclease Or amplifying an isolated copy of its complement, This results in a nick in the first and second recognition sequences of the first and second recognition sites. The 3 'ends of these nicked sites are extended to produce new target nucleic acid sequences. Or its complement, and the downstream copy of the target nucleic acid sequence or its phase. Complement is replaced during the course of the process; and           (h) when sufficient to produce a detectable amount of a separated copy of the target nucleic acid sequence Continuing the step (g) for a while.   The amplification step of the method according to the invention comprises the step of: Place at least one nick at the location, ie, form a cleavage. Long chain In addition to single-strand cleavage, the present invention provides for both strands of a double-stranded polynucleotide. It is also intended to form nicks simultaneously by nicking individually. I However, the simultaneous formation of such cleavages cannot occur at the same site in the target sequence. Absent. Cleavage at each strand highlights the target nucleic acid sequence between cleavage positions. That is, it must be substantially specified. Virtually semi-modified restriction end By combining a nuclease recognition site with the same type of restriction endonuclease , Good nick activity is obtained. Nicks can be added to semi-modified recognition sites Restriction endonucleases include AccI, AlwI, AvaI, BsaI, BsiHKCI, BsmI, Bso BI, BsrI, BstXI, DpnI, Fnu4HI, FokI, HincII, HindIII, HphI, MspI, NciI, Examples include, but are not limited to, NlaIV, NspI, Pf1MI, and Tth111I. Ni Particularly suitable for insertion of Bk are BsiHKCI and its recognition site. The position is semi-modified by incorporating dCTP (αS). Those skilled in the art will appreciate the invention Will come up with other means to nick your double-stranded polynucleotide . For example, the replication enzyme is a double Cleavage can be formed in the single strand of the strand polynucleotide. Or, DpnI certified Methylate a primer specific to the single strand of the recognition site using dam methylase be able to. Since DpnI cleaves only the methylated DNA strand, it is not repaired. By the nucleic acid monomer of the decoration and DpnI as the nick enzyme, the amplicon (ie, Separation and amplification of target nucleic acid sequences adjacent to nickable sites Copy) is generated.   Furthermore, it is also necessary to selectively nick one of the two nucleic acid strands at the recognition site. Modification of restriction endonucleases that alter enzymatic activity is also a feature of the present invention. Works. For example, an IIS type restriction endonuclease such as FokI is Reworking by site-directed mutagenesis to inactivate one of the cleavage centers So that the reworked enzyme cleaves only one of the two cleavage sites on the substrate. The nick enzyme according to the present invention can be obtained.   Deoxyribonucleoside modified with saturating amounts of deoxyribonucleoside triphosphate Since sidotriphosphate is used, the time for performing the amplification step depends on the activity of the polymerase and the temperature. Depends on the degree. Typically, the amplification time is about 60 minutes.   Alternatively, a method for amplifying a target nucleic acid sequence located within a single-stranded polynucleotide The above steps (g) and (h) as steps (d) and (e) respectively, a short separation method, steps (a)-(c) And that the execution procedure in each of the steps (g) and (h) is replaced at one place. And is implemented by First, in step (g), the `` third double-stranded polynucleotide The notation `` contact '' refers to `` contacting the double-stranded polynucleotide extension product '' in step (d). Yes ". Next, the notation of "step (g)" in step (h) is Replace with the notation of “step (d)” in (e). Amplification method described in this paragraph Is hereinafter referred to as “short amplification method, steps (a)-(e)”.   A third aspect of the invention relates to a method for determining the presence of a target nucleic acid sequence. Specifically The present invention determines the presence of a target nucleic acid sequence within a single-stranded polynucleotide. The method comprises the steps (a) to (h) described above and the following step (i);           (i) determining the presence of a detectable amount of the separated copy of the target nucleic acid sequence and detecting If the presence of a possible amount of separated copies of the target nucleic acid sequence is observed, Include additional steps to indicate that linked genotypes or organisms are present in the sample No.   In a preferred embodiment, the method of the invention comprises Cryptosporidium parvum. To determine the pathogenicity of Cryptosporidium parvum in potentially suspected samples Used for The pathogenic presence of Cryptosporidium parvum is determined by the steps (a)-(i) of the present invention. Is determined by subjecting the sample to the method described above. Cryptosporidium parvum nucleic acid Is detected by amplifying a part of the 18S rRNA gene of Cryptosporidium parvum. For detection, two amplification primers, P1 and P2, were used. P1 nucleo The nucleotide sequence is shown in SEQ ID NO: 2 and the nucleotide sequence of P2 is shown in SEQ ID NO: 3. Posted. DN, including visualization of stained gels containing reaction products fractionated by electrophoresis A. One or more different techniques for detection may be used to detect amplified target nucleic acid sequences. Used. In addition, probe extension using a detection system that uses radiolabels The Say method can also be used (see below). Such detection technology is a conventional technology. And are well known in the art.   In another preferred embodiment, the susceptibility of humans to breast cancer is determined. Specifically Contains genomic DNA obtained from female or male (ie, carrier) patients A sample is prepared for the target nucleic acid sequence. Use as a sampling source. Alleles deeply associated with genetic signs of breast and ovarian cancer To anneal to genomic DNA sequences that define mutation sites for diagnosis , Design primers. Specifically, mutations on chromosome 17q21.1 It is associated with a high prevalence of cancer and ovarian cancer. Heterozygous for such mutations Of women have an 85% chance of surviving breast cancer and 63% of ovarian cancer It is. Boyd et al., "A Human," the entire text of which is incorporated herein by reference. See BRCA1 Gene Knockout, “Nature 375: 541-542 (1995). The child was mapped in 1990 and encodes a protein of 1863 amino acids24 Exons. Hall et al., Incorporated herein by reference. Science 250: 1684-1689 (1990). The BRCA1 gene has already been isolated I have. Simar et al., Nature Genet, both of which are incorporated herein by reference. . 8: 392-398 (1994) and Miki et al., Science 266: 66-71 (1994). Germline mutations in BRCA1 have been identified in more than 80 families with these cancers I have. Futreal et al., Science 26, each of which is incorporated herein by reference. 6: 120-122 (1994), Friedman et al., Nature Genet. 8: 399-404 (1994), and And Schattuck-Eidens et al., A.J. Med. Ass. 273: 535-541 (1993) . Boyd et al (1995) found that families with a high prevalence of breast and / or ovarian cancer A1 sequence, i.e., two "A" nucleotides at position 2800 of exon 11 (AA₂₈₀₀) Deletion, which results in a stop codon at nucleotide 2820 Have reported that. Friedman et al (1994) used the same AA₂₈₀₀Reported a mutation in ing. The amplification method of the present invention is used for amplifying a known mutation as described above. Being If a mutant amplicon is detected, its presence will be Increases the risk of breast and / or ovarian cancer. For example, the known BRCA1 Sequence and deletion (AA₂₈₀₀) By using the two salts at the 3 'end. Group has two bases deleted (AA₂₈₀₀) A site having complementarity with the two bases upstream of One amplification primer is constructed. Using the known sequence for exon 11 of BRCA1 This creates a second amplification primer that hybridizes to the downstream complement. You. The first amplification primer constructed as described above has a mutation (AA₂₈₀₀) And just It can be completely hybridized. BRCA1 allele lacking this mutation Does not hybridize to the 3 'end of the first amplification primer and produces an amplification product. Absent. Conversely, in the BRCA1 gene with this mutation, the mutation increases to a detectable amount. It is width.   In another embodiment, the method of the present invention comprises the step of partially excluding 24 exons of the BRCA1 gene. Alternatively, it is used as a screening technique for detecting all mutations. example For example, following the sequence of the BRCA1 gene disclosed in the literature, its 3 'end is the target First to bind to the 3 'end of the target exon (or intron 3' to it) Design amplification primers. The second amplification primer according to the present invention is At the 3 'end of the complement of the target exon (or the complement of the intron 3' to it) It has a 3 'end designed to join. For example, forming a semi-modified recognition site U.S. Pat.No. 5,270,184, which relates to recognition sequences and recognition enzymes that can See description in column 10. These primers serve the purpose of a known BRCA1 gene. Design to define the target exon to be Incorporated herein by reference See also Nowak, Science 268: 1700-1701 (1995) That. The amplified exon (ie, the target nucleic acid sequence) is converted to the size of the breast cancer sign. To search for mutations at loci associated with, such as single-strand conformation polymorphism (SSCP) Can be separated electrophoretically using techniques well known in the art.   In another embodiment, the amplification primer is an Ataxia capillary vasodilator, a genetic disease. Defines a region of the ATM gene, known as a diagnostic clue to Zhang. Honcho Savitsky et al., Science 268: 1749-1753 (1995), incorporated herein by reference. Reports mapping and a portion of the single gene cDNA responsible for the disease. ing. The amplicon generated by the method of the present invention is a standard technology in the art. Familiar with the rapid determination of some of the human genotypes using Related human genes Knowledge of some of the types can also be used to identify mammals, including their ability to express specific disease features. Prompts you to determine your status. This finding has been implicated in cures that include preventive measures (ie, genetic counseling). It provides guidelines for rehabilitation work.   In another embodiment, the reverse transcriptase (RT), HIV-1 protein contained in the HIV-1 genome. Aase, or integrase, regulatory protein (Tat, REV, Nef), or Virion structural proteins and auxiliary proteins (Env, Gag, Vif, Vpr, Vpu) By amplifying a known nucleic acid sequence encoding such a structural protein, The method of the present invention for detecting a target nucleic acid sequence detects the presence of a retrovirus such as HIV-1 Used to In particular, by using the known sequence of HIV-1 RT, RT (RN A or any of its cDNAs) A first primer having a 3 'end that hybridizes to the 3' end of the Consisting of a second amplification primer having a 3 'end that hybridizes to the 3' end of complement, A pair of primers is a book Prepared according to the invention. The two amplification primers used in the method of the present invention Defines the region of the nucleic acid sequence and requires about 16 or more complementary bases per primer Since the partial sequence of the end opposite to the target nucleic acid sequence of interest is Again, the method of the present invention works well. In this embodiment, each amplification primer Is a restriction endonuclease that can nick a semi-modified recognition site At its 5 'end. Deoxyribonucleoside triphosphate And the presence of the modified deoxyribonucleoside triphosphate described in the above steps (a) to (i). In the presence, the amplification primers described above and nuclei obtained from a sample considered to contain HIV-1 By using an acid, the method of the present invention amplifies HIV-1 RT in a sample and Is detected. The detection method of the present invention described above can be used for DNA synthesis of AZT, DDC, DDI, 3TC, etc. The rapid response seen with pharmacotherapy with anti-RT agents that function as chain stoppers during growth The present invention can also be applied to amplification of a target nucleic acid sequence of HIV-1 such as rapidly mutating RT.   In another embodiment, the method for determining the presence of a target nucleic acid sequence comprises the step (i) ) As step (f), a short amplification method, and a method having a configuration added to steps (a) to (e). detection The latter method for determining the presence of a possible amount of a target nucleic acid sequence is hereinafter referred to as the “short determination method, (A)-(f) ".   Another aspect of the present invention relates to a kit comprising components for performing the method of the present invention. And this kit:           (a) Dehydration to produce an essential first product from the complement of the template A polymerizing enzyme capable of polymerizing xyribonucleoside triphosphates, A polymerizable enzyme that can be           (b) a pair of amplification primers including a first amplification primer and a second amplification primer The first amplification primer is a large The 3 'end of the target nucleic acid sequence located at a position other than the end of the polynucleotide Nicks can be added to the complementary 3 'end and the semi-modified first recognition site Has a 5 'end with a first restriction sequence for a first restriction endonuclease, Further, the second amplification primer has complementarity with the 3 ′ end of the complement of the target nucleic acid sequence. 2 'restriction with a 3' end and a nicked semi-modified second recognition site A pair of amplified having a 5 'end with a second restriction sequence for an endonuclease Width primer;           (c) Nicking a substantially semi-modified restriction endonuclease recognition site One or two restriction endonucleases that can be           (d) deoxyribonucleoside triphosphate; and           (e) a modified deoxyribonucleoside triphosphate.   Whether the first recognition sequence and the second recognition sequence are identical or different, Are included within the scope of the present invention. In addition, the kit described above has an RNA-dependent It may further comprise a DNA polymerase. In addition, the yeast used in the kit Kits may contain sufficient buffers and ions to efficiently maintain elemental activity. , Within the scope of the present invention. In the kit of the present invention, the enzyme is freeze-dried. It is provided in a state or in a stabilizing solution.   In a preferred embodiment, it is placed in a buffer solution stabilized with glycerol, And provided at -20 ° C. The kit includes AMV reverse transcriptase, ribonuclease H, Bst DNA polymerase, Bs iHKCI and bovine serum albumin, magnesium chloride, other salts, Manito And an enzyme dilution buffer containing trehalose and trehalose. A person skilled in the art If other Enzymes, enzyme subunits and fragments thereof are also useful in kits according to the invention. You will notice that it can be used. Deoxyribonucleic acid used in the kit of the present invention Reoside triphosphates include 2'-deoxyguanosine 5'-triphosphate, 2'-deoxyadenoate Nosin 5'-triphosphate, thymidine 5'-triphosphate, and 2'-deoxycytidine 5'-triphosphate Includes 3 of the 4 substances in the group consisting of acid but used in this kit One unmodified deoxyribonucleoside triphosphate is the number of its modified Used in one form. Modified deoxyribonucleoside used in the present invention Triphosphate is as described above. Preferred modified deoxyribonucleic acid Osidotriphosphate is 2'-deoxyguanosine 5'-0- (l-thiotriphosphate), 2'-deoxy Siadenosine 5'-0- (1-thiotriphosphate), thymidine 5'-0- (1-thiotriphosphate), and 2′-deoxycytidine 5′-0- (1-thiotriphosphate). In another embodiment, the chloride Add tramethylammonium or tetraethylammonium chloride to the reaction components Can be   In a preferred kit of the invention, the following are standard 1.4 ml screwed It is placed in a gasketed microcentrifuge tube with a cap. 10 × buffer solution strike Pack is 350mM K ・ PO_Four(pH 7.6). In the second tube The 10X stock of other reagents was 102 mM magnesium chloride, 7.0 mM Tris-HCl (pH 7. 9), 34 mM potassium chloride, 7.0 mM dithiothreitol, 20% (w / v) maltitol, And 0.5 ml of a solution containing 13.4% (w / v) trehalose. 5mM dGTP, 5m 0.5 ml of a 10 × stock solution consisting of MdATP, 5 mM TTP, and 14 mM dCTP (αS) was prepared. A third tube is filled with deoxyribonucleoside triphosphate. -20 ° C Units of AMV reverse transcriptase placed in stabilized glycerol solution maintained at room temperature , 24 simple E. of rank coli ribonuclease H, 800 units of Bst DNA polymerase, 15,000 units 10 × stock solution consisting of BsiHKCI in position 2 and 7.0 mg / ml bovine serum albumin The protein is placed in a fourth tube charged with 0.5 ml of. Prepared in this way Further, the kit of the present invention is provided with reagents sufficient for performing a conventional amplification reaction 100 times. The user of the kit should be able to use the test containing or suspected of containing the target nucleic acid sequence Prepare the fee. Each of the two primers (eg, SEQ ID NOs: 2 and 3; 0.5 μM) Each target binds at least about 16 bases near the 3 'end of each primer It has a part. Near the 5 'end of each of the two primers, BsiH The sequence specifying the chain of the recognition site for KCI, 5'-CTCGGG-3 '(ie, the recognition sequence ). For the heat denaturation step in the method of the present invention, a buffer and a deoxyribonucleic acid Reoside triphosphate, a sample nucleic acid and a primer are used in combination. For example Utilizes a denaturation step that includes a 2 minute incubation at 100 ° C. For example, Following incubation at a suitable annealing temperature such as 2 minutes at about 60 ° C And add the remaining reaction components and allow the reaction to proceed, for example, at about 60 ° C for 60 minutes. Let me go.                   Separation and / or amplification procedures   The separation / amplification procedure performed by the method of the present invention is shown in FIGS. 1a and 1b. Illustrative purpose In the following, an embodiment relating to a target nucleic acid sequence within single-stranded polynucleotide 1 will be described. (FIG. 1a). First, deduced from the target nucleic acid sequence (eg, RNA or DNA) The sequence data was designed using two primers, (+) primer 2 and (-) primer 5. Used for Each primer separates and amplifies towards its 5 'end Semi repair of double strands formed between Restriction endonuclease for restriction endonucleases that nick decorative DNA It contains a protease recognition sequence. (+) 3 'end of primer 2 is within polynucleotide 1 And a target binding region that shows a sequence complementary to the 3 'end of the target nucleic acid sequence at (-) The 3 ′ end of the primer 5 is a sequence found in the complementary (+) strand 3, A target binding sequence that is complementary to the 3 'end of the complement of the target nucleic acid sequence in Otide 1. Includes joint areas. The target binding region of (+) primer 2 in polynucleotide 1 and ( -) The complement of the target binding region of primer 5 And the portion of the polynucleotide between the primer sequences to be amplified Defines the range of the target nucleic acid sequence.   If the target nucleic acid sequence is double-stranded, the amplification procedure may be, for example, polynucleotide. Starting from a separation step (not shown) by denaturation. For example, standard in the art Denaturation can be achieved by heating or changing the pH using any suitable technique. In a preferred embodiment, two primers, (+) primer 2 and (-) primer Immer 5 and deoxyribonucleoside triphosphate (at least one of which is repaired) Reaction) in the presence of a mixture containing buffering agents and salts. The chains are separated by heating. Among other things, the reaction is By increasing the temperature to 0 ° C., the chains are separated. Particularly preferably, at 100 ° C. for 2 minutes During the incubation, the strands are separated. Includes target polynucleotide Appropriate so that (+) primer 2 anneals to the resulting polynucleotide 1 The temperature is reduced to, for example, 60 ° C. (step I in FIG. 1a, not shown).   In step II of FIG. 1a, where polynucleotide 1 is an RNA molecule, the template-specific Polymerases capable of polymerizing nucleic acids, such as For example, RNA-dependent DNA polymerases such as reverse transcriptase By extension of the first amplification primer using nucleotide 1 and (+) primer 2, The complementary (+) strand 3 is synthesized. When polynucleotide 1 is RNA, reverse transcriptase Element catalyzes the synthesis of the complementary (+) strand 3 (in this case, the cDNA) It is an enzyme. This reaction involves the reaction of deoxyribonucleoside triphosphate with at least one Proceeds in the presence of the modified deoxyribonucleoside triphosphate. DNA port Extension of (+) Primer 2 by a polymerizing enzyme such as remelase or reverse transcriptase Form the complementary (+) chain 3. Hybridization of complementary (+) strand 3 to polynucleotide 1 Digestion is partially double (ie, partially double-stranded) polynucleotides. Produce product 4, which is an otide. Complementary (+)-strand 3 is the one known in the art. Is separated from polynucleotide 1 by the method. Polynucleotide 1 is RNA When it is a molecule, it is preferably an RNA polynucleic acid involved in an RNA / DNA duplex. Separation is performed using an enzyme that selectively degrades Reotide 1, which is illustrated in FIG. The broken line indicates the polynucleotide 1 'partially degraded by I. RNA / Selectively acts on the RNA strand of DNA duplex and has ribonuclease H activity Enzymes containing the obtained enzymes are preferred enzymes. This step involves ribonuclease H Catalyzed by active reverse transcriptase or another ribonuclease H enzyme You. Preferred enzymes are E. coli. coli ribonuclease H enzyme. Effectively split the chain Other methods for separating include heating the mixture and adjusting the pH. Partially Regardless of whether or not to go through the steps for the unraveled polynucleotide 1 ', As shown in FIG. IV, the complementary (+) strand 3 becomes a functional single strand.   In step V, the complementary (+) strand 3 is the second amplification primer, (-) Contact with primer 5. This (−) primer 5 is used as the 3′-binding region of (−) primer 5. Sequence that defines one end of the target nucleic acid sequence, which is substantially identical to the sequence of the region. Anneal to the region of the complementary (+) strand 3 containing the complementary sequence. As defined herein Thus, the substitution of uracil for thymine and ribose for deoxyribose The main difference, that is, RNA and DNA sequences with substantially the same information, Sequences are identical, and constitute copies of each other.   In step VI, the complementary (+) strand 3 as a template and at least one of Using the decorated deoxyribonucleoside triphosphate, the (-) primer 5 It is extended. This extension reaction has a partial duplex with the complementary (+) strand 3. To produce a modified (−) strand 6 involved in the polynucleotide 7 (step VI).   Polynucleotide 7, which is partially double-stranded, contains two incompletely modified strands. No. The 5 'end of each strand of the (+) strand 3, which is complementary to the modified (-) strand 6, is an unmodified restriction endonuclease. It is provided by an unmodified primer sequence containing a creatase recognition sequence. (-) The 5 'end of strand 6 is with the (-) primer 5 and the 5' end of the complementary (+) strand 3 is with the (+) primer. Provided by Limer 2. Therefore, a partially double-stranded polynucleotide The single-stranded treated restriction endonuclease recognition site at Otide 7 is Since the unmodified (+) primer 2 is involved in the formation, it is substantially semi-modified. This is the recognized recognition site. Figures 1a and 1b show nickable restriction endonucleators The zeta recognition sites are represented as juxtaposed boxes with different patterns. Juxtaposed box phases The boundaries of each other are cleavage sites.   In step VII, the partially double-stranded polynucleotide 7 The semi-modified restriction endonuclease recognition site at is a single-stranded unmodified restriction endonuclease. Cleavage of nuclease sequences by restriction endonucleases (e.g., BsiHKCI) Nick is put in. This nick creates an upstream (+) strand 8 and a downstream (+) strand 9 And both strands are annealed to the unnicked modified (-) strand 6 I have. The upstream (+) strand 8 is another polynucleotide that is complementary to the modified (-) strand 6. It has a free 3 'end available for synthesis with the tide chain.   In step VIII, the 3 'end of the upstream (+) strand 8 is complementary to the modified (-) strand 6 It is extended to form a modified (+) strand 10. In this step, the (+) chain 9 Removed from Annealing the (+) strand 10 modified to the (-) strand 6 yields non-target DNA A double-stranded polynucleotide fragment 11 substantially containing no is generated. Double-stranded polynucle The non-target DNA at Reotide Fragment 11 mainly consists of the two introduced restriction endonucleases. It is a nuclease recognition site. Therefore, the double-stranded polynucleotide fragment 11 A double-stranded form of a separated copy of the target nucleic acid sequence, suitable for amplification by the method of the invention . Double stranded polynucleotide fragment 11 is substantially semi-modified and may be nicked It has two possible restriction endonuclease recognition sites, which are It is located at each end. Nicking double-stranded polynucleotide fragment 11 These two restriction endonuclease recognition sites that can be It may be different.   According to FIG. 1b, double-stranded polynucleotide fragment 11 is a semi-modified restriction endonucleic acid. Nicking process at the rease recognition site (step IX) and the 3 'generated by the nick The target nucleic acid sequence or its complement (step The strand of the downstream chain containing X) is displaced. Specifically, a new (+) chain 15 is It is formed by extending the upstream (+) strand 8. With the synthesis of the new (+) strand 15, The displaced (+) strand 12 is removed. Although not shown in FIG.1b, the substituted (+) strand 12 (-) Binds to primer 5. By extending (-) primer 5, a new (-) strand 1 6 (not shown). As synthesis proceeds, a new (+) strand 15 is substituted for the substituted (-) strand 13. Hybridize to form a new duplex 17, as shown in step XIa. new Duplex 17 is one regenerated restriction endonuclease that can contain a nick Includes a protease recognition site. Nicking the site cleaves a new (+) strand 15 A stream (+) strand 8 and a small downstream (+) strand 19 (step XIIa) are formed. In process XIIIa, upstream (+) Extending strand 8 produces another copy of the new (+) strand 15, while a small lower strand The current (+) chain 19 is coming off. In step XIVa, when the extension reaction is completed, the substituted (-) strand A complete copy of the new (+) strand 15 hybridized to 13 is generated, which , A new duplex 17 regenerates. The new duplex 17 is prepared in steps XIIa, XIIIa and XIVa Will be added again. The displaced small downstream (+) strand 19 (step XVa) was (Step XVIa). New (-) strand by extension of (-) primer 5 Sixteen copies were generated and a small one using the sequence of (-) primer 5 as a template. Extending the downstream (+) strand 19 produces a copy of the displaced (+) strand 12 (step XVIIa). As synthesis proceeds, the (+) strand 12 substituted for the new (-) strand 16 hybridizes, Thus, a new copy of the duplex 18 is formed that functions as a substrate in step XIIb. A similar reaction system is used for operations involving the (-) strand 13 replacing the upstream (-) strand 14 (engineering). About X).   In step XIb, by extending the upstream (-) chain 14, a new (-) chain 16 is generated. The annealed substituted (−) strand 13 is removed. The substituted (-) strand 13 is the (+) primer 2 Hybridized to Then, the (+) primer 2 was extended to hybridize to the displaced (−) strand 13. A new (+) strand 15 is created, which creates a copy of the new duplex 17 (not shown) You. In step XIIb, the new (-) strand 16 involved in the new duplex 18 is nicked. This produces an upstream (−) strand 14 and a small downstream (−) strand 20. Upstream (-) chain 14 Elongation generates a new (-) strand 16 and removes a small downstream (-) strand 20 ( Step XIIIb). In step XIVb, the new (−) strand 16 was replaced with the substituted (+) strand 12 and hybridized. Completes the synthesis of the (-) strand 16 copy because a new duplex 18 copy is generated . The new duplex 18 is then reapplied to steps XIIb, XIIIb and XIVb. The displaced small downstream (-) strand 20 (step XVb) hybridizes to (+) primer 2 (Step XVIb). Extension of (+) primer 2 generates new copy of (+) strand 15 And using the (+) primer 2 as a template to extend the small downstream (-) strand 20 Thus, a substituted (−) chain 13 is generated (Step XVIIb). New (+) strand 15 is replaced by (-) strand 13 As it hybridizes, a new copy of duplex 17 is generated, which is passed to step XIIa. Sent. These steps will be repeated while the amplification process is in progress. You.   (+) Primer 2 and (-) Primer 5 used in this method are generally Reotide has a length of about 40. Targets for (+) primer 2 and (-) primer 5 The binding site is highly stringent, resulting from standard reaction conditions. Expresses complementarity under hybridization conditions so that it can bind to the target template Should be an array.Experimental example             1. Amplification of RNA target polynucleotide   All drugs were prepared at reagent level. Moloney mouse leukemia virus Reverse transcriptase and Superscript II transcriptase Purchased from Rosie (Rockville, MD). T3 RNA polymerase Ambion Maxiscript (Austin, Texas) mbion Maxiscript). All other enzymes are molecular Sourced from Biology Resources, Milwaukee, Wisconsin . Synthesize polynucleotides and use synthetic genetics (Sy Gel filtration by nthetic Genetics (San Diego, CA).   RNA templates were prepared using the Ambion Maxiscript kit according to the manufacturer's instructions. Made. In short, the RNA template was prepared by transcription of about 2 μg of plasmid pCPV4.7. Made. Plasmid pCPV4.7 contains the 18S rRNA gene from Cryptosporidium parvum Contains. (This plasmid is used for disease control in Atlanta, Georgia. Courtesy of Norman Pieniazek of the Center ) This plasmid contains the T3 RNA polymerase promoter in close proximity to the 18S rRNA gene. And the promoter is suitably integrated to express the gene. I have. This plasmid was digested by restriction endonuclease digestion with PvuII. And T3 RNA polymerase and Ambion Maxiscript kit to Transcribed in vivo. The reaction product is washed with ribonucleic acid to completely remove DNA. Washed twice with nuclease-free deoxyribonuclease I. Transcript, Known standard on agarose gel And quantified by comparison. 1.5 μg of pure transcript was obtained. Final concentration of reaction components The degree is usually 35 mM potassium phosphate (pH 7.6), 1.4 mM dCTP (αS), 0.5 mM dATP, 0.5 mM mM dGTP, 0.5 mM dTTP, 0.7 mg / ml bovine serum albumin, 10.2 mM magnesium chloride , 0.7 mM Tris-HCl (pH 7.9), 3.4 mM potassium chloride, 0.7 mM dithiothreitol, 2% maltitol, 1.34% trehalose, 4-8 units of AMV reverse transcriptase, 0.12-0 .24 units of E. coli ribonuclease H, 8 units of Bst DNA polymerase, 150 units BsiHKCI and the variable amount of RNA template described in each experiment described below. Make the final reaction volume 50 μl. In these samples, primers P1 and P2 were also 0.5 μM each. (See below). In addition, this reaction component has a concentration of 1-100 mM. Optionally contains tetramethylammonium chloride or tetraethylammonium chloride .   P1 nucleotide sequence, 5'-ACCCCATCCAATGCATGTc / tcgggTCGTAGTCTTAACCAT-3 ' As SEQ ID NO: 2. [Primer P1 is the (+) primer of FIGS. -2. ] Nucleotide sequence of P2, 5'-CGATTCGGCTCCAGACTTc / tcgggTGCT GAAGGAGTAAGG-3 ′ was described as SEQ ID NO: 3. [Primer P2 is shown in FIGS. 1a and 1 This corresponds to (-) primer 5 in b. In the sequence of P1 and P2 described above, BsiHKCI The sequence corresponding to the restriction endonuclease recognition sequence in lower case, and , The slash is the restriction endonuclease cleavage site or the site where the nick enters It is. The sequence from 3 'to each restriction endonuclease recognition sequence is the target of P1 and P2. It is a binding region.   40 μl sample containing phosphate buffer, nucleotides, primers and template Was prepared using concentrated reagents. In order to denature the template, the sample is , Heated to 100 ° C. The remaining reaction components, ie, bovine serum album Min, salt Magnesium chloride, Tris-hydrochloric acid (pH 7.9), potassium chloride, dithiothreitol, Litol, trehalose, reverse transcriptase, Bst DNA polymerase, BsiHKCI and The sample was allowed to stand for 2 minutes before adding the enzyme mixture containing Cooled to 50 ° C. In a one-step protocol, samples were incubated at 50 ° C for 60 minutes. In the two-step protocol, samples are first incubated at 37 ° C for 30 minutes. And then incubate at 50 ° C. for 30 minutes.   See Walker et al., Nucl. Acids Res., Which is incorporated herein by reference. 20 (7): 1961-1969 (1992). Detected. First, the Cryptosporidi of 5'-AGAGATTGGAGGTTG-3 'described in SEQ ID NO: 4 The probe for detecting um parvum was radiolabeled as follows. 10 pmol C.I. Parvum detection probe, 35 μCi [γ³²P] ATP (6000Ci / mmol, NEN Research Pro Kutsu, Boston, Mass.), 30 units of polynucleotide kinase , 50 mM Tris-HCl, pH 7.2, 1 mM magnesium chloride, 5 mM dithiothreitol, A total volume of 10 μl of 0.1 mM spermidine-hydrochloric acid was incubated at 37 ° C. for 30 minutes. Was. The reaction was stopped by heating to 100 ° C. in 5 minutes. Next, radioactively labeled C . Parvum detection probe contains 10 mM dATP, dCTP, dGTP and TTP each; 5 μl of 350 mM potassium phosphate, pH 7.6; 1.5 μl of 200 mM magnesium chloride; A solution containing water was added to bring the final volume of the mixture to 50 μl. And radiation The labeled detection probe was subjected to a detection assay.   5 μl of the reaction mixture containing the radiolabeled detection probe and amplicon mix The combination with 5 μl of the mixture was heated to 95 ° C. for 2 minutes and then 37 ° C. And incubate for 2 minutes Was. At this point, 1 μl of exo^-DNA polymerer Ze (Klenow fragment; 10 units / μl) was added. Incubate this sample at 37 ° C for 15 minutes. Incubate and add 95% formaldehyde, 10 mM EDTA, 1 mM dithiotre Itol, 0.05% (w / v) bromophenol blue, and 0.05% (w / v) xylene The reaction was stopped with 11 μl of the solution containing the anole. Next, the sample was heated at 95 ° C for 2 minutes. And electrophoresed on a 10% denaturing polyacrylamide gel. Therefore, it was analyzed. These gels are run on standard autoradiographs Applied to detection technology. In this embodiment according to the method of the present invention, 57 (total length) ) And the production of 38 (nick) nucleotide products. 2. Amplification of RNA target polynucleotide using various polymerization agents   Various polymerization agents were used to amplify the RNA target nucleic acid sequence of the present invention. That The results are shown in FIG. The reaction was repeated as described in section 1 above.   An RNA transcript for use as a template in the method of the present invention is Prepared as above using mid pCPV4.7 and T3 RNA polymerase. Multiple anti Each sample of available RNA template is approximately 10⁷Molecules. RNA Transcript copy number is determined spectrophotometrically using the enriched template stock and a transcript size of 1750 bases It was determined analytically at 260 nm. A portion of the template was placed in a total volume of 250 μl at 37 ° C. for 60 minutes. 25 μg ribonucleic acid in 10 mM Tris-HCl, pH 8.5, 50 mM potassium chloride Treated with ZeA. The RNA template sample was used without further treatment.   For the amplification reaction, 35 mM potassium phosphate (pH 7.6), 1.4 mM dCTP (α S), 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM TTP, 0.7 mg / ml bovine serum albumin, 10.2 m M magnesium chloride, 0.7 mM Tris-hydrochloric acid (pH 7.9), 3.4 mM potassium chloride, 0.7 mM Dichi Otreitol, 2% maltitol, 1.34% trehalose, 0.5 μM P1 primer -, 0.5 μM P2 primer, 150 units of BsiHKCI, and various Type and amount of reverse transcriptase were used. In a reaction using 4 units of AMV reverse transcriptase, 0 .12 units of E. coli ribonuclease H was also used (FIG. 2, lanes 1-4). Figure Reactions fractionated in lanes 5-8 of No. 2 contained 200 units of MMLV reverse transcriptase (MMLV reverse transcriptase has an intrinsic ribonuclease H activity). Fig. 2 The reaction products shown in Tables 9-12 were generated using 200 units of Superscript II ( Superscript II is a proprietary MMLV reverse transcriptase lacking endogenous ribonuclease H activity Prime). The reaction products shown in lanes 2, 6 and 10 in FIG. Without exposure to ZeA, the RNA template⁶Generated using Lanes 3 and 7 The reaction products shown in and 11 were treated with ribonuclease A as described above to give Ten⁶Was generated using a single molecule. Reaction products shown in lanes 4, 8 and 12 in FIG. Was generated using approximately 500 linearized DNA molecules as a negative control. Lane 1, The "product" at 5 and 9 was produced without any form of the target nucleic acid sequence. And thus also serve as negative controls.   25 μl of sample (control DNA, RNA template or water) and bring the total volume to 40 μl 15 μl phosphate buffer containing deoxyribonucleotides and primers The reaction was started by mixing the liquids. Incubate the reaction at 100 ° C for 2 minutes. I did it. The remaining reaction components, namely the enzymes described above, bovine serum albumin, salt Magnesium chloride, Tris-hydrochloric acid (pH 7.9), potassium chloride Enzyme mixture containing uranium, dithiothreitol, maltitol and trehalose The reaction mixture was cooled to 37 ° C. for 2 minutes before adding 10 μl of the product. So And incubate the samples at 37 ° C for 30 minutes, then at 50 ° C for 30 minutes did. All reactions were stopped by heating to 100 ° C. for 2 minutes. Earlier Amplicons were detected by a modified probe extension assay. Specific reverse transcriptase Reverse transcriptase and ribonucleic acid that produce intrinsic or exogenous activity In lanes 2 and 6 of FIG. Complicons were observed. All samples treated with ribonuclease A were tested. No available amplicon was produced. Similarly, reactions lacking the target nucleic acid sequence And a reaction using a DNA plasmid template, produce detectable amplicons. Did not reach.                             3. Balanced amplification   This example substantially demonstrates the equilibrium amplification of an RNA template according to the method of the present invention. Things. For this reaction, 35 mM potassium phosphate (pH 7.6), 1.4 mM dCTP (αS) , 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM TTP, 0.7 mg / ml bovine serum albumin, 0.7 mM T ris-hydrochloric acid (pH 7.9), 3.4 mM potassium chloride, 0.7 mM dithiothreitol, 2% mulch Toll, 1.34% trehalose, 8 units of Bst DNA polymerase, 150 units of Bsi HKCI and RNA template were used. In these samples, the primers P1 and P2 were also used in each It was included at a concentration of 5 μM. The reaction containing the RNA template is added to the Cryptosporidium parvum 425 pg of RNA transcription template expressed from the 18S rRNA gene was added. These reactions Are shown in lanes 2 to 9 of FIG. In FIG. 3, lane 1 “product” is cast Lacks mold This was due to a control reaction. The concentration of magnesium chloride is shown in FIG. For each enzyme that was evaluated, adjust it in the range of 7.5 to 12.5 mM (final reaction concentration). It was adjusted. AMV-RT reactions contained 4 units (Figure 3, lanes 1 and 2) or 8 units (Figure 3, The enzyme from lane 9) was added. MMLV-RT-catalyzed reaction contains 200 units of reverse transcriptase But added E. coli ribonuclease H was not added (FIG. 3, lanes 3-5). ). Superscript II RT-catalyzed reactions include 200 units of reverse transcriptase and 0.24 units E. coli ribonuclease H was added (FIG. 3, lanes 6-8).   Controls containing 25 μl of RNA template (FIG. 3, lanes 2-9) or 15 μl of water ( FIG. 3, lane 1) and deoxyribonucleotides for a total volume of 40 μl And the reaction was started by mixing 15 μl of phosphate buffer containing primers. . The reaction was incubated at 100 ° C. for 2 minutes. Split into lanes 3 to 9 in FIG. The applied reaction was cooled to 50 ° C. in 2 minutes; the product of lanes 1-2 in FIG. The resulting reaction was cooled to 37 ° C. in 2 minutes. Then the remaining reactants (top Enzyme described, bovine serum albumin, magnesium chloride, Tris-HCl (pH 7.9), salt Potassium iodide, dithiothreitol, maltitol and trehalose) 10 μl of the enzyme mixture was added. Samples to be tested in lanes 3 to 9 in FIG. , Incubated for 60 minutes; the samples analyzed in lanes 1-2 in FIG. For 30 minutes then at 50 ° C. for 30 minutes. In 2 minutes, heat up to 100 ° C Heating stopped all reactions. According to the probe extension assay described above, Amplicon was detected. Figure 3 shows that amplicon formation is template-dependent. And that amplicons were observed under virtually all equilibrium conditions. are doing. Substantially equilibrium conditions are substantially Contains constant temperature and constant reactants. Separated copy of target nucleic acid sequence and its complement Can be nicked in a polynucleotide fragment containing Separation or modification of polynucleotides required before incorporating the enzyme recognition site Temperature changes may occur during the first few cycles to promote sex However, a substantially constant temperature is maintained during each amplification cycle. Real The amount of amplicon produced under equilibrium conditions (Figure 3, lanes 3-9) was determined by AMV- The amount of amplicon observed in the two-step temperature reaction using RT (Fig. 3, lane 2) It was comparable.                       4. Sensitivity of the method of the invention   The nature of the method of the invention for amplifying variable amounts of target nucleic acid sequences to detectable levels Noh was tested. For this reaction, 35 mM potassium phosphate, pH 7.6, 10.2 mM magnesium chloride was used. Cium, 1.4 mM dCTP (αS), 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM TTP, 0.7 mg / ml bovine Serum albumin, 0.7 mM Tris-HCl (pH 7.9), 3.4 mM potassium chloride, 0.7 mM dithio Threitol, 2% maltitol, 1.34% trehalose, 8 units of BstDNA poly Merase, 150 units of BsiHKCI, 0.24 units of E. coli. coli ribonuclease H, and Using 8 units of AMV reverse transcriptase, the final reaction volume for each reaction was 50 μl. This These samples contained primers P1 and P2 at a concentration of 0.5 μM each, and As described, RNA transcript of 18S rRNA gene from Cryptosporidium parvum In the form of 10⁰~Ten^FiveRNA target nucleic acid sequence copies were also included. RNA copy The number was determined as described above. The concentrated template was washed with 10 mM Tris-HCl, pH 8.5. And diluted with 50 mM potassium chloride.   25 μl RNA template (FIG. 4, lanes 2-7) or 15 μl water Control (FIG. 4, lane 1) and deoxylysin to a total volume of 40 μl. By mixing 15 μl of phosphate buffer containing primers with bonucleotides, The reaction was started. The reaction was incubated at 100 ° C. for 2 minutes. The rest of the reaction Ingredients (enzyme, bovine serum albumin, magnesium chloride, Tris-HCl (pH 7 .9), potassium chloride, dithiothreitol, maltitol and trehalose) The reaction was cooled to 50 ° C. for 2 minutes before adding 10 μl of the enzyme mixture containing Rejected. Amplicons were detected by the probe extension assay described above. Figure The results shown in Figure 4 indicate that amplicons were observed under virtually all equilibrium conditions, In addition, the present invention provides an RNA target polynucleotide having a small copy number of 100 copies. Even when applied to the method of (a), detectable amplicon was observed (Fig. 5).   Although the invention has been described with respect to particular embodiments, those skilled in the art will appreciate that the invention can be modified. It is thought to be correct and come up with a change. Therefore, only the limitations indicated in the attached claims Should be added to the present invention.

[Procedure for Amendment] [Date of Submission] April 20, 1999 (April 20, 1999) [Content of Amendment] Claims (1) A method for separating a copy of a target nucleic acid sequence contained in a single-stranded polynucleotide and suitable for use in amplifying a target nucleic acid sequence, comprising the following steps: a) preparing a reaction mixture, i.e .; (i) a sample, i.e., a sample containing a target nucleic acid sequence at a position other than the end of the single-stranded polynucleotide, or containing the target nucleic acid sequence. (Ii) Polymerization of deoxyribonucleoside triphosphate by extending the hybridized primer using the same or different first and second polymerases, ie, the single-stranded polynucleotide as a template. The first polymerizing enzyme and the hybridized primer are extended to polymerize deoxyribonucleoside triphosphate, and (Iii) a mixture of deoxyribonucleoside triphosphates, including modified deoxyribonucleoside triphosphates, if the nucleic acid is DNA; (iv) hybridizing to the target nucleic acid sequence and hybridizing thereto A first amplification primer forming a soybean primer, comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, wherein the 3 ′ end of the first amplification primer is located at the 3 ′ end of the target nucleic acid sequence. And the 5 'end of the first amplification primer is capable of nicking one strand of a double-stranded semi-modified recognition site for a first restriction endonuclease. A first amplification primer comprising a first recognition sequence for a first restriction endonuclease; and (v) a second amplification primer comprising oligodeoxyribonucleotides having 3 'and 5' ends. A width primer, wherein the 3 'end of the second amplification primer is complementary to and hybridizes to the 3' end of the complement of the target nucleic acid sequence, and the second amplification primer At the 5 'end is provided with a second recognition sequence for a second restriction endonuclease, wherein the second restriction endonuclease is capable of recognizing one of the double-stranded semi-modified recognition sites that it recognizes. Preparing a reaction mixture comprising a second amplification primer that is a restriction endonuclease capable of nicking the strand; (b) providing a ribonuclease enzyme that separates the double-stranded nucleic acid in the reaction mixture into single strands; Allowing the reaction mixture to act; and (c) allowing the reaction mixture to stand for a time sufficient to effect hybridization, primer extension, and nicking, thereby providing a separate copy of the target nucleic acid sequence. A double-stranded polynucleotide extension product suitable for amplification, comprising an isolated copy of the complement of the target nucleic acid sequence, i.e., at the opposite end, the first and second restriction endonucleases, respectively. Producing a double-stranded polynucleotide extension product defined by the first and second restriction sites nicked by the target nucleic acid sequence contained in the single-stranded polynucleotide. How to separate copies. (2) The method according to claim 1, wherein the reaction mixture is an aqueous or substantially aqueous medium. (3) The method according to claim 1 or 2, wherein the ribonuclease enzyme is ribonuclease H. (4) A method for separating a copy of a target nucleic acid sequence contained in a single-stranded polynucleotide and suitable for use in amplifying a target nucleic acid sequence, comprising the following steps: The first amplification primer is hybridized to the 3 'end of the target nucleic acid sequence contained in the strand polynucleotide, that is, to form a hybridized first amplification primer, the target nucleic acid sequence contained in the single-stranded polynucleotide Hybridization of a first amplification primer to the 3 ′ end, wherein the first amplification primer comprises an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end, and the 3 ′ end of the first amplification primer Is complementary to the 3 'end of the target nucleic acid sequence, and the 5' end of the first amplification primer comprises a first recognition sequence for a first restriction endonuclease. Further, the first restriction endonuclease is an endonuclease capable of nicking one strand of a double-stranded semi-modified recognition site that it recognizes, thereby comprising a single-stranded polynucleotide. (B) extending the hybridized first amplification primer from its 3 ′ end, ie, having a complementarity with the single-stranded polynucleotide. The deoxyribonucleoside triphosphate was modified using the target nucleic acid sequence as a template to form a first double-stranded polynucleotide having the modified first polynucleotide extension product attached thereto. In the presence of deoxyribonucleoside triphosphate, and a primary polymerizing enzyme that polymerizes deoxyribonucleoside triphosphate, its 3 ′ Extending the first amplification primer hybridized from the end; (c) separating the single-stranded polynucleotide from the modified first polynucleotide extension product by an enzyme; (d) modifying the modified first polynucleotide extension product Hybridization of the second amplification primer to the internal site of the second amplification primer, i.e., hybridization of the second amplification primer to the internal site of the modified first polynucleotide extension product to form a hybridized second amplification primer. Wherein the second amplification primer comprises an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end, and the 3 ′ end of the second amplification primer is 3 ′ of the complement of the target nucleic acid sequence. Is complementary to and hybridizes to the end, and the 5 'end of the second amplification primer has a second restriction endonucleic acid. An enzyme capable of nicking one strand of a double-stranded semi-modified recognition site that the second restriction endonuclease recognizes. A nuclease, whereby a second amplification primer is hybridized to an internal site of the modified first polynucleotide extension product; (e) extending the hybridized second amplification primer from its 3 ′ end, Deoxyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate to form a second double-stranded polynucleotide comprising the modified second polynucleotide extension product hybridized to the modified first polynucleotide extension product. Polymerizing deoxyribonucleoside triphosphates in the presence of a polynucleotide template, and Extending the hybridized second amplification primer from its 3 ′ end with a reaction mixture containing a second polymerase that displaces the strand, wherein the modified second polynucleotide extension product comprises one A modified first polynucleotide comprising a copy of the target nucleic acid sequence separated from the main-stranded polynucleotide and located at the opposite end between the second recognition sequence and the complement of the first recognition sequence; The complement of the first recognition sequence in the nucleotide extension product and the first recognition sequence in the second polynucleotide extension product forms a double-stranded, semi-modified recognition site for the first recognition endonuclease. Thereby extending the hybridized second amplification primer from its 3 'end; and (f) one of the double-stranded, semi-modified, recognition sites for the first recognition endonuclease. On the chain of The first restriction endonuclease, the deoxyribonucleoside triphosphate, the modified deoxyribonucleoside triphosphate, and two primers for the first recognition endonuclease in the presence of the second polymerase. Nicking the semi-modified recognition site of a strand into one strand, thereby extending the nicked 3 ′ end, thereby modifying the modified first primary site downstream thereof. Any portion of the polynucleotide extension product can be replaced and the first and second recognition sites can be nicked by the first and second restriction endonucleases, respectively. The portion of the target nucleic acid sequence that has hybridized to the separated copy of the complement of the target nucleic acid sequence, as defined by the opposite end. A method of forming a third double-stranded polynucleotide containing a copy, comprising the step, to separate the copies of the target nucleic acid sequence contained in a single-stranded polynucleotide, wherein the. (5) The method according to claim 4, wherein the method is carried out in the presence of at least one substance selected from the group consisting of mannitol, trehalose, tetramethylammonium chloride and tetraethylammonium chloride. (6) The method according to any one of claims 1 to 5, wherein the target nucleic acid sequence is RNA. (7) The method according to any one of claims 4 to 6, wherein the step (c) includes introducing ribonuclease activity. (8) The method according to claim 7, wherein the ribonuclease activity is ribonuclease H activity. (9) The first restriction endonuclease is AccI, AlwI, AvaI, BsaI, BsiHKCI, BsmI, BsoBI, BsrI, BstXI, DpnI, Fnu4HI, FokI, HincII, HindIII, HphI, MspI, NciI, NlaIV, NspI, 9. The method according to claim 1, wherein the method is selected from the group consisting of PflMI and Tth111I. (10) The method according to claim 9, wherein the first restriction endonuclease is BsiHKCI. (11) The second restriction endonuclease is AccI, AlwI, AvaI, BsaI, BsiHKCI, BsmI, BsoBI, BsrI, BstXI, DpnI, Fnu4HI, FokI, HincII, HindIII, HphI, MspI, NciI, NlaIV, NspI, 9. The method according to claim 1, wherein the method is selected from the group consisting of PflMI and Tth111I. (12) The method according to claim 11, wherein the second restriction endonuclease is BsiHKCI. (13) The method according to any one of claims 1 to 8, wherein the first restriction endonuclease and the second restriction endonuclease are the same endonuclease. (14) The method according to any one of claims 1 to 13, wherein the nickable first recognition site and the nickable second recognition site are the same. (15) the deoxyribonucleoside triphosphate is 2'-deoxyguanosine 5'-0- (1-thiotriphosphate), 2'-deoxyadenosine 5'-0- (1-thiotriphosphate), thymidine 5'-0- (1-thiotriphosphate), 2'-deoxycytidine 5'-0- (1-thiotriphosphate), 2'-deoxyuridine 5'-triphosphate, 5-methyldeoxy 15. The method according to any one of claims 1 to 14, wherein the method is selected from the group consisting of cytidine 5'-triphosphate and 7-deaza-2'-deoxyguanosine 5'-triphosphate. (16) The method according to any one of claims 1 to 15, wherein the first polymerizing enzyme is a polymerase. (17) The method according to any one of claims 1 to 15, wherein the first polymerizing enzyme is an enzyme having reverse transcriptase activity. (18) The method according to any one of claims 1 to 17, wherein the second polymerase is a DNA polymerase. (19) The DNA polymerase is Bst DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Tf1 DNA polymerase, E. coli. coli DNA polymerase, E. coli. coli DNA polymerase Klenow fragment and exo ^- 19. The method according to claim 18, which is a DNA polymerase selected from the group consisting of E. coli DNA polymerase Klenow fragment. (20) The method according to claim 18 or 19, wherein the DNA polymerase is Bst DNA polymerase. (21) The method according to any one of claims 1 to 15, wherein the first synthase and the second synthase are the same synthase. (22) A kit for separating a copy of a target nucleic acid sequence contained in a single-stranded polynucleotide, the kit comprising: (a) for producing a first product essential from a complement of a template; A polymerization enzyme capable of polymerizing deoxyribonucleoside triphosphate and capable of strand displacement; (b) a pair of amplification primers including a first amplification primer and a second amplification primer, wherein the first amplification primer is A 3 'end that is complementary to the 3' end of the target nucleic acid sequence located at a location other than the end of the large polynucleotide, and a first restriction endonuclease that can nick the semi-modified first recognition site And the second amplification primer has a 3 'end that is complementary to the 3' end of the complement of the target nucleic acid sequence, and a semi-modified To the second recognition site A pair of amplification primers having a 5 'end with a second restriction sequence for a second restriction endonuclease capable of receiving a restriction; (c) a nick at the substantially semi-modified restriction endonuclease recognition site. (D) deoxyribonucleoside triphosphate; (e) a modified deoxyribonucleoside triphosphate; and (f) an enzyme having ribonuclease activity. A kit for separating a copy of a target nucleic acid sequence contained in a single-stranded polynucleotide, the kit comprising: (23) The polymerizing enzyme is avian myeloblastosis virus reverse transcriptase, Moloney murine leukemia virus reverse transcriptase, superscript II, Bst DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, E. coli. DNA polymerase Klenow fragment and exo ^- E. 23. The kit according to claim 22, wherein the kit is selected from the group consisting of E. coli DNA polymerase Klenow fragment. (24) The restriction endonuclease is AccI, AlwI, AvaI, BsaI, BsiHKCI, BsmI, BsoBI, BsrI, BstXI, DpnI, Fnu4HI, FokI, HincII, HindIII, HphI, MspI, NciI, NlaIV, NspI, PflMI and PflMI. 24. The kit according to claim 22 or 23 selected from the group consisting of: (25) The kit according to claim 22 or 23, wherein the restriction endonuclease is BsiHKCI. (26) A method for amplifying a copy of a target nucleic acid sequence contained in a single-stranded polynucleotide, comprising the following steps: (a) preparing a reaction mixture, that is, (i) a sample, That is, a sample containing a target nucleic acid sequence at a position other than the end of the single-stranded polynucleotide, or a sample considered to contain the target nucleic acid sequence; (ii) a first and a second polymerase which are the same or different from each other That is, using the single-stranded polynucleotide as a template, a first polymerizing enzyme that polymerizes deoxyribonucleoside triphosphate by extending a hybridized primer, and a deoxyribonucleoside by extending a hybridized primer. A second polymerizing enzyme that polymerizes triphosphate and further displaces the strand if the target nucleic acid is DNA; A mixture of deoxyribonucleoside triphosphates containing a decorated deoxyribonucleoside triphosphate; (iv) a first amplification primer that hybridizes to a target nucleic acid sequence and forms a primer hybridized therewith, wherein the 3 ′ end and 5 ′ An oligodeoxyribonucleotide with a 'end', the 3 'end of the first amplification primer is complementary to the 3' end of the target nucleic acid sequence, and the 5 'end of the first amplification primer is A first amplification comprising a first recognition sequence for the first restriction endonuclease capable of nicking one strand of a double-stranded semi-modified recognition site for the first restriction endonuclease A primer: and (v) a second amplification primer comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, wherein the 3 ′ end of the second amplification primer is the target nucleic acid. Complementary to and hybridizes to the 3 'end of the complement of the sequence, and the 5' end of the second amplification primer carries a second recognition sequence for a second restriction endonuclease. A second amplification primer, wherein the second restriction endonuclease is a restriction endonuclease that is capable of nicking one strand of a double-stranded semi-modified recognition site that it recognizes. (B) reacting the reaction mixture with a ribonuclease enzyme that separates double-stranded nucleic acids in the reaction mixture into single strands; (c) performing hybridization, primer extension, and nicking. The reaction mixture is allowed to stand for a time sufficient to perform, thereby providing a double-stranded plasmid suitable for amplification, comprising a separate copy of the target nucleic acid sequence and a separate copy of the complement of the target nucleic acid sequence. A oligonucleotide extension product, i.e., a double-stranded poly defined by a first and a second restriction site nicked by the first and second restriction endonucleases at opposite ends, respectively. (D) an enzyme capable of amplifying an isolated copy of the target nucleic acid sequence or its complement, ie having DNA polymerase activity and displacing the strand, deoxyribonucleoside triphosphates and modifications Contacting the double-stranded polynucleotide extension product with the first restriction endonuclease and the second restriction endonuclease in the presence of the resulting mixture of deoxyribonucleoside triphosphates to separate the target nucleic acid sequence or its complement. Amplifying the copy, whereby the first recognition site and the second Nicking the first recognition sequence and the second recognition sequence of the recognition site, extending the 3 'end of these nicked sites to produce a new target nucleic acid sequence or its complement, and Displacing the downstream copy of the nucleic acid sequence or its complement during the process; and (e) performing step (d) for a time sufficient to produce a detectable amount of the isolated copy of the target nucleic acid sequence. A method for amplifying a copy of a target nucleic acid sequence contained in a single-stranded polynucleotide, comprising the steps of continuing. (27) A method for amplifying a copy of a target nucleic acid sequence contained in a single-stranded polynucleotide, comprising the following steps: (a) adding a 3′-terminal to the 3 ′ end of the target nucleic acid sequence contained in the single-stranded polynucleotide; Hybridization of the first amplification primer, i.e., hybridization of the first amplification primer to the 3 'end of the target nucleic acid sequence contained in the single-stranded polynucleotide to form a hybridized first amplification primer. Wherein the first amplification primer comprises an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, and the 3 ′ end of the first amplification primer is complementary to the 3 ′ end of the target nucleic acid sequence. And the 5 'end of the first amplification primer comprises a first recognition sequence for a first restriction endonuclease, and further, the first restriction endonuclease has An endonuclease capable of nicking one strand of a double-stranded semi-modified recognition site for recognition, thereby allowing the first amplification at the 3 'end of the target nucleic acid sequence contained in the single-stranded polynucleotide. (B) extending the hybridized first amplification primer from its 3 'end, i.e., the modified first amplification polynucleotide that is complementary to and bound to the single-stranded polynucleotide. Using the target nucleic acid sequence as a template, a deoxyribonucleoside triphosphate, a modified deoxyribonucleoside triphosphate, and a deoxyribonucleoside triphosphate to form a first double-stranded polynucleotide having one polynucleotide extension product The first amplification primer hybridized from the 3 'end of the first amplification enzyme (C) separating the single-stranded polynucleotide from the modified first polynucleotide extension product by an enzyme; (d) placing a second amplification primer at an internal site of the modified first polynucleotide extension product. Hybridization of the second amplification primer to an internal site of the modified first polynucleotide extension product to form a hybridized, ie, hybridized, second amplification primer, wherein the second amplification primer comprises: The primer comprises an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end, and the 3 ′ end of the second amplification primer is complementary to the 3 ′ end of the complement of the target nucleic acid sequence. And hybridizing thereto, and the 5 'end of the second amplification primer has a second recognition sequence for a second restriction endonuclease; The second restriction endonuclease is an endonuclease capable of nicking one strand of a double-stranded, semi-modified recognition site that it recognizes, whereby the modified first polynucleotide extension product (E) extending the hybridized second amplification primer from its 3 ′ end, i.e., hybridizing to the modified first polynucleotide extension product. Polymerize deoxyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate, and deoxyribonucleoside triphosphate in the presence of the polynucleotide template to form a second double-stranded polynucleotide containing the two polynucleotide extension products Reaction mixture containing a second polymerase that displaces the strand Extending the hybridized second amplification primer from the 3 ′ end thereof, wherein the modified second polynucleotide extension product is separated from the single-stranded polynucleotide and the second recognition sequence A copy of the target nucleic acid sequence located at the opposite end between the complement of the first recognition sequence and the first recognition sequence and a second polynucleotide in a modified first polynucleotide extension product The complement of the first recognition sequence in the extension product forms a double-stranded, semi-modified recognition site for the first recognition endonuclease, thereby forming a hybridized second amplification primer. Extending from its 3 'end; (f) nicking one strand of the double-stranded, semi-modified recognition site for the first recognition endonuclease, ie, the first restriction endonuclease; One of the double-stranded, semi-modified recognition sites for the first recognition endonuclease in the presence of oxyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate, and the second synthase. Nicking the strand, thereby extending the nicked 3 ′ end, thereby displacing any portion of the modified first polynucleotide extension product downstream thereof; And the target nucleic acid defined by opposing ends by the first and second recognition sites that can be nicked by the first and second restriction endonucleases, respectively. Forming a third double-stranded polynucleotide comprising an isolated copy of the target nucleic acid sequence hybridized to an isolated copy of the complement of the sequence; ( g) in the presence of a mixture of deoxyribonucleoside triphosphates and modified deoxyribonucleoside triphosphates, an enzyme with DNA polymerase activity capable of amplifying an isolated copy of the target nucleic acid sequence or its complement, i.e. displacing the strand. Contacting the third double-stranded polynucleotide with the first restriction endonuclease and the second restriction endonuclease to amplify a separated copy of the target nucleic acid sequence or its complement, thereby The first recognition sequence and the second recognition sequence of the one recognition site and the second recognition site are nicked, and the 3 'ends of these nicked sites are extended to form a new target nucleic acid sequence or its complement. And replacing the downstream copy of the target nucleic acid sequence or its complement during the process; and (h) detecting Continuing the step (g) for a sufficient amount of time to produce as many separate copies of the target nucleic acid sequence as possible, comprising: copying the target nucleic acid sequence contained in the single-stranded polynucleotide. How to amplify. (28) The method according to claim 27, wherein the steps (g) and (h) proceed under substantially isothermal conditions. (29) The method according to claim 28, wherein the isothermal condition is an isothermal condition at a temperature of 37 ° C to 60 ° C. (30) The method according to claim 29, wherein the isothermal condition is an isothermal condition at a temperature of 60 ° C. (31) A method for determining the presence of a target nucleic acid sequence contained in a single-stranded polynucleotide, comprising the following steps: (a) preparing a reaction mixture, that is, (i) a sample, That is, a sample containing a target nucleic acid sequence at a position other than the end of the single-stranded polynucleotide, or a sample considered to contain the target nucleic acid sequence; (ii) a first and a second polymerase which are the same or different from each other That is, using the single-stranded polynucleotide as a template, a first polymerizing enzyme that polymerizes deoxyribonucleoside triphosphate by extending a hybridized primer, and a deoxyribonucleoside by extending a hybridized primer. A second polymerizing enzyme that polymerizes triphosphate and further displaces the strand if the target nucleic acid is DNA; (iii) modification A mixture of deoxyribonucleoside triphosphates containing a modified deoxyribonucleoside triphosphate; (iv) a first amplification primer that hybridizes to a target nucleic acid sequence and forms a primer hybridized thereto, wherein the 3 ′ end and 5 ′ An oligodeoxyribonucleotide with an end, the 3 'end of the first amplification primer is complementary to the 3' end of the target nucleic acid sequence, and the 5 'end of the first amplification primer is A first amplification primer comprising a first recognition sequence for the first restriction endonuclease capable of nicking one strand of a double-stranded semi-modified recognition site for the first restriction endonuclease And (v) a second amplification primer comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, wherein the 3 ′ end of the second amplification primer is the target nucleic acid sequence. Is complementary to and hybridizes to the 3 ′ end of the complement of the second amplification primer, and the 5 ′ end of the second amplification primer comprises a second recognition sequence for a second restriction endonuclease. And a second amplification primer, wherein the second restriction endonuclease is a restriction endonuclease that is capable of nicking one strand of a double-stranded semi-modified recognition site that it recognizes. Preparing a reaction mixture; (b) subjecting the reaction mixture to a ribonuclease enzyme that separates double-stranded nucleic acids in the reaction mixture into single strands; (c) performing hybridization, primer extension, and nicking The reaction mixture is allowed to stand for a period of time sufficient to allow a double stranded plasmid, suitable for amplification, containing a separate copy of the target nucleic acid sequence and a separate copy of the complement of the target nucleic acid sequence. A nucleotide extension product, i.e., a double-stranded polynucleotide defined by a first and a second restriction site nicked by the first and second restriction endonucleases at opposite ends, respectively. (D) an enzyme capable of amplifying an isolated copy of the target nucleic acid sequence or its complement, ie having DNA polymerase activity and displacing the strand, deoxyribonucleoside triphosphate and the modified Contacting the double-stranded polynucleotide extension product with the first restriction endonuclease and the second restriction endonuclease in the presence of a mixture of deoxyribonucleoside triphosphates results in a separate copy of the target nucleic acid sequence or its complement. Amplifying the first recognition site and the second recognition site. Nicking the first recognition sequence and the second recognition sequence of the recognition site, extending the 3 'end of these nicked sites to produce a new target nucleic acid sequence or its complement, and Displacing the downstream copy of the nucleic acid sequence or its complement during the process; (e) continuing step (d) for a time sufficient to produce a detectable amount of the isolated copy of the target nucleic acid sequence And (f) determining the presence of a detectable amount of the isolated copy of the target nucleic acid sequence, the method comprising detecting the presence of the target nucleic acid sequence contained in the single-stranded polynucleotide. (32) A method for determining the presence of a target nucleic acid sequence contained in a single-stranded polynucleotide, comprising the following steps: (a) 3 ′ end of the target nucleic acid sequence contained in a single-stranded polynucleotide The first amplification primer is hybridized to the first amplification primer, that is, the first amplification primer is hybridized to the 3 ′ end of the target nucleic acid sequence contained in the single-stranded polynucleotide to form a hybridized first amplification primer. The first amplification primer includes an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, and the 3 ′ end of the first amplification primer is located relative to the 3 ′ end of the target nucleic acid sequence. Complementary, and the 5 'end of the first amplification primer is provided with a first recognition sequence for a first restriction endonuclease, and further, the first restriction endonuclease is It is an endonuclease that can nick one strand of the double-stranded semi-modified recognition site that it recognizes, thereby providing a 3′-end at the 3 ′ end of the target nucleic acid sequence contained in the single-stranded polynucleotide. (B) a modification wherein the hybridized first amplification primer extends from its 3 'end, ie, is complementary to and bound to the single-stranded polynucleotide. Using a target nucleic acid sequence as a template, a deoxyribonucleoside triphosphate, a modified deoxyribonucleoside triphosphate, and a deoxyribonucleoside triphosphate are used to form a first double-stranded polynucleotide having a modified first polynucleotide extension product. First amplification primer hybridized from its 3 'end in the presence of the first polymerase that polymerizes phosphate (C) separating the single-stranded polynucleotide from the modified first polynucleotide extension product by an enzyme; and (d) placing a second amplification primer at an internal site of the modified first polynucleotide extension product. Hybridization of a second amplification primer to an internal site of the modified first polynucleotide extension product to form a hybridized, ie, hybridized, second amplification primer, wherein the second amplification primer is Comprises an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end, and the 3 ′ end of the second amplification primer is complementary to the 3 ′ end of the complement of the target nucleic acid sequence, Also hybridized thereto, further, the 5 'end of the second amplification primer has a second recognition sequence for a second restriction endonuclease, The second restriction endonuclease is an endonuclease capable of nicking one strand of a double-stranded semi-modified recognition site that it recognizes, whereby the modified first polynucleotide is (E) the hybridized second amplification primer is extended from its 3 ′ end, ie, the modified hybridized to the modified first polynucleotide extension product. A deoxyribonucleoside triphosphate, a modified deoxyribonucleoside triphosphate, and a deoxyribonucleoside triphosphate in the presence of the polynucleotide template to form a second double-stranded polynucleotide comprising the modified second polynucleotide extension product. And a reaction involving a second polymerase that displaces the chain An extension of the hybridized second amplification primer from its 3 ′ end with the compound, wherein the modified second polynucleotide extension product is separated from a single-stranded polynucleotide and the second recognition A copy of the target nucleic acid sequence located at the opposite end between the sequence and the complement of the first recognition sequence, and the first recognition sequence and the second in the modified first polynucleotide extension product. The complement of the first recognition sequence in the polynucleotide extension product forms a double-stranded, semi-modified recognition site for the first recognition endonuclease, whereby the hybridized second amplification Extending a primer from its 3 ′ end; (f) nicking one strand of the double-stranded, semi-modified recognition site for the first recognition endonuclease, ie, the first restriction endonuclease. Nuclear A deoxyribonucleoside triphosphate, a modified deoxyribonucleoside triphosphate, and one of the double-stranded semi-modified recognition sites for the first recognition endonuclease in the presence of the second polymerase. Nicking the strand, thereby extending the nicked 3 ′ end, thereby displacing any portion of the modified first polynucleotide extension product downstream thereof; And the target nucleic acid defined by opposite ends by the first and second recognition sites nickable by the first and second restriction endonucleases, respectively. Form a third double-stranded polynucleotide containing an isolated copy of the target nucleic acid sequence hybridized to an isolated copy of the complement of the sequence (G) an enzyme having DNA polymerase activity capable of amplifying an isolated copy of the target nucleic acid sequence or its complement, ie, displacing the strand, a mixture of deoxyribonucleoside triphosphates and modified deoxyribonucleoside triphosphates; Contacting the third double-stranded polynucleotide in the presence with the first restriction endonuclease and the second restriction endonuclease to amplify a separated copy of the target nucleic acid sequence or its complement, thereby The first and second recognition sites of the first and second recognition sites are nicked and the 3 'ends of these nicked sites are extended to provide a new target nucleic acid sequence or Complementation occurs and displaces a downstream copy of the target nucleic acid sequence or its complement during the process; (h) detection Continuing step (g) for a time sufficient to produce an effective amount of the isolated copy of the target nucleic acid sequence; and (i) determining the presence of a detectable amount of the isolated copy of the target nucleic acid sequence; A method for determining the presence of a target nucleic acid sequence contained in a single-stranded polynucleotide, comprising: (33) The method according to claim 32, wherein the steps (g) and (h) proceed under substantially isothermal conditions. (34) The method according to claim 33, wherein the isothermal condition is an isothermal condition at a temperature of 37 ° C to 60 ° C. (35) The method according to claim 34, wherein the isothermal condition is an isothermal condition at a temperature of 60 ° C.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventor Mueller, Rainhold             United States 53226 Wisconsin               Wow Wattsa North 86 Street             G 1930 (72) Brassac, Michael, El.             United States 53226 Wisconsin               Wow Wattsa West Blue Mount             Do 11205 Apartment 103 (72) Inventor Wilcos, Richard, K.             United States 53151 Wisconsin               New Berlin Coachlight Dora             Eve 1715 Be

Claims (1)

[Claims] 1. A method for separating copies of a target nucleic acid sequence found in a single-stranded polynucleotide, wherein the separated copies are suitable for use for amplification of a target nucleic acid sequence, the method comprising: Preparing a mixture, the reaction mixture comprising: (i) a sample containing, or believed to contain, the target nucleic acid sequence at a position other than the terminus in the single-stranded polynucleotide; Using a single strand as a template, a first polymerizing enzyme capable of polymerizing deoxyribonucleoside triphosphate by extending a hybridized primer, and deoxyribonucleoside triphosphate by extending a hybridized primer. A second polymerizing enzyme capable of polymerizing a nucleic acid, and further capable of displacing a strand if the target nucleic acid is DNA, (Iii) a mixture of deoxyribonucleoside triphosphates containing modified deoxyribonucleoside triphosphates; (iv) A) a first amplification primer capable of hybridizing to a target nucleic acid sequence and forming a primer hybridized thereto, the first amplification primer comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end; Is complementary to the 3 'end of the target nucleic acid sequence, and the 5' end of the first amplification primer is a double-stranded, semi-modified recognition site for a first restriction endonuclease. A first amplification primer having a first recognition sequence for the first restriction endonuclease capable of nicking one strand; and (v) an amplifying primer having 3 ′ and 5 ′ ends. A second amplification primer comprising a Godeoxyribonucleotide, wherein the 3 'end of the second amplification primer is complementary to and hybridizes to the 3' end of the complement of the target nucleic acid sequence. The 5 'end of the second amplification primer has a second recognition sequence for a second restriction endonuclease, which endonuclease is capable of recognizing the double-stranded semi-modified recognition thereof. A second amplification primer that is a restriction endonuclease capable of nicking one strand of the site; (b) reacting the double-stranded nucleic acid in the reaction mixture with a single-stranded nucleic acid; And (c) leaving the reaction mixture for a time sufficient to effect hybridization, primer extension, and nicking, thereby providing said target nucleus. A double-stranded polynucleotide extension product is generated, comprising a separate copy of the sequence and a separate copy of the complement of the target nucleic acid sequence, suitable for amplification, wherein the double-stranded polynucleotide extension product is located at the opposite end. Wherein the nicks for each of the first and second restriction endonucleases are defined by a first restriction site and a second restriction site, respectively, comprising the steps of: A method for separating copies. 2. A method for separating copies of a target nucleic acid sequence found in a single-stranded polynucleotide, wherein the separated copies are suitable for amplification of a target nucleic acid sequence, the method comprising: (a) a single-stranded polynucleotide; The first amplification primer hybridizes to the 3 'end of the target nucleic acid sequence within, i.e., the 3' end of the target nucleic acid sequence within the single-stranded polynucleotide to form a hybridized first amplification primer. To the first amplification primer, wherein the first amplification primer comprises an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, and the 3 ′ end of the first amplification primer is the target nucleic acid. Complementary to the 3 'end of the sequence, the 5' end of the first amplification primer has a first recognition sequence for a first restriction endonuclease, and Is capable of nicking one strand of the double-stranded semi-modified recognition site that it recognizes; (b) extending the hybridized first amplification primer from its 3 ′ end. The target nucleic acid sequence to form a first double-stranded polynucleotide that is complementary to the single-stranded polynucleotide and has a modified first polynucleotide extension product attached thereto. Was used as a template to hybridize from its 3 'end in the presence of deoxyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate, and a first polymerizing enzyme capable of polymerizing deoxyribonucleoside triphosphate. Extending the first amplification primer; (c) separating the single-stranded polynucleotide from the modified first polynucleotide extension product; A second amplification primer is hybridized to an internal site of the first polynucleotide extension product, i.e., to form a hybridized second amplification primer, to an internal site of the modified first polynucleotide extension product. A hybridization of a second amplification primer, wherein the second amplification primer comprises an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, and the 3 ′ end of the second amplification primer is the target nucleic acid sequence. Complementary to and capable of hybridizing to the 3 'end of the complement, the 5' end of the second amplification primer has a second recognition sequence for a second restriction endonuclease. The endonuclease is capable of nicking one strand of the double-stranded semi-modified recognition site that it recognizes. (E) extending a hybridized second amplification primer from its 3 ′ end, ie, a second two primers comprising a modified second polynucleotide extension product hybridized to the modified first polynucleotide extension product; Deoxyribonucleoside triphosphates, modified deoxyribonucleoside triphosphates, and deoxyribonucleoside triphosphates can be polymerized in the presence of a polynucleotide template to form a strand polynucleotide, and can displace the strand. Extension of a hybridized second amplification primer from its 3 'end with a reaction mixture comprising a mixture of possible second polymerases, wherein the modified second polynucleotide extension product is a single-stranded polynucleotide. And the opposite end between the second recognition sequence and the complement of the first recognition sequence Wherein the complement of the first recognition sequence in the modified first polynucleotide extension product and the first recognition sequence in the second polynucleotide extension product comprises a copy of the target nucleic acid sequence located at Forming a double-stranded semi-modified recognition site for a nuclease; and (f) on one strand of the double-stranded semi-modified recognition site for the first recognition endonuclease. Nicked, i.e., the first restriction endonuclease, the deoxyribonucleoside triphosphate, the modified deoxyribonucleoside triphosphate, and the two for the first recognition endonuclease in the presence of the second polymerase. Nicking the semi-modified recognition site of the strand into one strand, whereby the nicked 3 ′ end is extended, Thus, any portion of the modified first polynucleotide extension product downstream thereof can be replaced and the first and second nicks for each of the first and second restriction endonucleases can be nicked. Forming a third double-stranded polynucleotide comprising a separated copy of the target nucleic acid sequence hybridized to a separated copy of the complement of the target nucleic acid sequence, defined at opposite ends by the recognition site of A method for separating copies of a target nucleic acid sequence found in a single-stranded polynucleotide, comprising: 3. The method according to claim 1, wherein the reaction mixture is an aqueous or substantially aqueous medium. 4. 3. The method according to claim 1, wherein the target nucleic acid sequence is RNA. 5. 3. The method of claim 2, wherein said step is performed in the presence of at least one of mannitol, trehalose, tetramethylammonium chloride, and tetraethylammonium chloride. 6. The method according to claim 4, wherein the separation step (c) is performed using an enzyme. 7. 7. The method according to claim 6, wherein said separating step (c) comprises introducing ribonuclease activity. 8. The method according to claim 7, wherein the ribonuclease activity is ribonuclease H activity. 9. 4. The method according to claim 3, wherein said separating step (c) comprises heating. Ten. The first restriction endonuclease is AccL, AlwI, AvaI, BsaI, BsiHKCI, BsmI, BsoBI, BsrI, BstXI, DpnI, Fnu4HI, FokI, HincII, HindIII, HphI, MspI, NciI, NlaIV, NspI, PflMI and Tth111. The method of claim 3 wherein the claim is selected from the group consisting of: 11． 11. The method according to claim 10, wherein said first restriction endonuclease is BsiHKCI. 12． The second restriction endonuclease is AccI, AlwI, AvaI, BsaI, BsiHKCI, Bsml, BsoBI, BsrI, BstXI, DpnI, Fnu4HI, FokI, HincII, HindIII, HphI, MspI, NciI, NlaIV, NspI, PflI and Tthll. The method of claim 3 wherein the claim is selected from the group consisting of: 13. 13. The method according to claim 12, wherein said second restriction endonuclease is BsiHKCI. 14. 4. The method according to claim 3, wherein said first restriction endonuclease and said second restriction endonuclease are the same. 15． 15. The method according to claim 14, wherein the nickable first recognition site and the nickable second recognition site are the same. 16． The modified deoxyribonucleoside triphosphate is 2′-deoxyguanosine 5′-0- (1-thiotriphosphate), 2′-deoxyadenosine 5′-0- (1-thiotriphosphate), thymidine 5 '-0- (1-thiotriphosphate), 2'-deoxycytidine 5'-0- (1-thiotriphosphate), 2'-deoxyuridine 5'-triphosphate, 5-methyldeoxycytidine 5 4. The method according to claim 3, wherein the method is selected from the group consisting of '-triphosphate or 7-deaza-2'-deoxyguanosine 5'-triphosphate. 17． The method according to claim 3, wherein the first polymerase is a polymerase. 18． 18. The method according to claim 17, wherein the first polymerase has a reverse transcriptase activity. 19. The method according to claim 3, wherein the second polymerase is a DNA polymerase. 20. The second polymerase is Bst DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, E. coli. coli DNA polymerase, E. coli. coli DNA polymerase Klenow fragment and exo ^- E. 20. The method according to claim 19, wherein the method is selected from the group consisting of E. coli DNA polymerase Klenow fragment. twenty one. 21. The method according to claim 20, wherein said second polymerase is Bst DNA polymerase. twenty two. The method according to claim 3, wherein the first and second polymerases are the same. twenty three. A kit comprising elements for performing the method of the invention, the kit comprising: (a) polymerizing deoxyribonucleoside triphosphate to produce a first product consisting essentially of the complement of the template. (B) a pair of amplification primers including a first amplification primer and a second amplification primer, wherein the first amplification primer is a large polynucleotide. A 3 'end with complementarity to the 3' end of the target nucleic acid sequence located at a position other than the end, and a first for a first restriction endonuclease capable of nicking the semi-modified first recognition site. A 5′-end having a restriction sequence, and wherein the second amplification primer has a 3′-end having complementarity to the 3′-end of the complement of the target nucleic acid sequence, and a semi-modified second recognition site Nick A pair of amplification primers having a 5 'end with a second restriction sequence for a second restriction endonuclease; (c) nicking a substantially semi-modified restriction endonuclease recognition site. A kit comprising, as possible, one or two restriction endonucleases; (d) a deoxyribonucleoside triphosphate; and (e) a modified deoxyribonucleoside triphosphate. twenty four. The polymerizing enzyme is avian myeloblastosis virus reverse transcriptase, Moloney murine leukemia virus reverse transcriptase, superscript II, Bst DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, E. coli. coli DNA polymerase Klenow fragment and exo ^- E. 24. The kit according to claim 23, wherein the kit is selected from the group consisting of E. coli DNA polymerase Klenow fragment. twenty five. The restriction endonuclease comprises AccI, AlwI, AvaI, BsaI, BsiHKCI, BsmI, BsoBI, BsrI, BstXI, DpnI, Fnu4HI, FokI, HincII, HindIII, HphI, MspI, NciI, NlaIV, NspI, PflMI and Tth111. 24. The kit according to claim 23, selected from: 26. 26. The kit according to claim 25, wherein the restriction endonuclease is BsiHKCI. 27. A method for amplifying a copy of a target nucleic acid sequence found in a single-stranded polynucleotide, comprising: (a) preparing a reaction mixture, the reaction mixture comprising: (i) a single-stranded polynucleotide A sample containing the target nucleic acid sequence at a position other than the terminal end thereof or containing the target nucleic acid sequence; (ii) using a single-stranded polynucleotide as a template to extend the hybridized primer First polymerizing enzyme capable of polymerizing deoxyribonucleoside triphosphate, and deoxyribonucleoside triphosphate can be polymerized by extending a hybridized primer, and further, if the target nucleic acid is DNA, displacing the strand. A second polymerizing enzyme, wherein the first polymerizing enzyme can optionally be the same as the second polymerizing enzyme. (Iii) a mixture of deoxyribonucleoside triphosphates containing modified deoxyribonucleoside triphosphates; (iv) capable of hybridizing to a target nucleic acid sequence and forming a primer hybridized thereto. A first amplification primer comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, the 3 ′ end of the first amplification primer being complementary to the 3 ′ end of the target nucleic acid sequence. The 5 'end of the first amplification primer can nick one strand of a double-stranded semi-modified recognition site for a first restriction endonuclease. A first amplification primer having one recognition sequence; and (v) a second amplification primer comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end. The 3 'end of the second amplification primer is complementary to the 3' end of the complement of the target nucleic acid sequence and is capable of hybridizing thereto, and the 5 'end of the second amplification primer Has a second recognition sequence for a second restriction endonuclease, wherein the endonuclease is capable of nicking one strand of a double-stranded semi-modified recognition site that it recognizes. (B) applying the reaction mixture to a means for separating double-stranded nucleic acids in the reaction mixture into single strands; (c) hybridization, primer extension, And allowing the reaction mixture to stand for a period of time sufficient to effect nicking, thereby comprising two separate copies of the target nucleic acid sequence and the complement of the target nucleic acid sequence, suitable for amplification. A double-stranded polynucleotide extension product is produced, which comprises, at opposite ends, a first restriction site into which a nick for each of the first and second restriction endonucleases is nicked. (D) an enzyme capable of amplifying an isolated copy of the target nucleic acid sequence or its complement, ie having DNA polymerase activity and displacing the strand, deoxyribonucleoside triphosphate and modified By contacting the double-stranded polynucleotide extension product with the first restriction endonuclease and the second restriction endonuclease in the presence of a mixture of deoxyribonucleoside triphosphates, an isolated copy of the target nucleic acid sequence or its complement To thereby amplify the first and second of the first and second recognition sites. The recognition sequence is nicked, the 3 'ends of these nicked sites are extended, resulting in a new target nucleic acid sequence or its complement, and the downstream copy of the target nucleic acid sequence or its complement is processed. And (e) continuing step (d) for a period of time sufficient to produce a detectable amount of a separated copy of the target nucleic acid sequence, comprising the steps of: A method for amplifying a copy of an observed target nucleic acid sequence. 28. A method for amplifying a copy of a target nucleic acid sequence found in a single-stranded polynucleotide, comprising: (a) first amplifying at the 3 'end of the target nucleic acid sequence within the single-stranded polynucleotide; Hybridizing the primers, i.e., hybridizing the first amplification primer to the 3 'end of the target nucleic acid sequence within the single-stranded polynucleotide to form a hybridized first amplification primer, The first amplification primer includes an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, and the 3 ′ end of the first amplification primer is complementary to the 3 ′ end of the target nucleic acid sequence, The 5 'end of the first amplification primer has a first recognition sequence for a first restriction endonuclease, wherein the endonuclease is capable of recognizing a double-stranded, semi-modified recognition site. (B) extending the hybridized first amplification primer from its 3 ′ end, ie, being complementary to the single-stranded polynucleotide, and The target nucleic acid sequence is used as a template to form a deoxyribonucleoside triphosphate, modified deoxyribonucleoside triphosphate to form a first double-stranded polynucleotide having a modified first polynucleotide extension product attached thereto. Extending a hybridized first amplification primer from its 3 ′ end in the presence of a first polymerase capable of polymerizing phosphate and deoxyribonucleoside triphosphate; (c) a modified first polynucleotide (D) separating the single-stranded polynucleotide from the extension product; Hybridizing an amplification primer, i.e., hybridizing a second amplification primer to an internal site of the modified first polynucleotide extension product to form a hybridized second amplification primer, wherein the second amplification primer comprises: The second amplification primer includes an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, and the 3 ′ end of the second amplification primer is complementary to the 3 ′ end of the complement of the target nucleic acid sequence, It is also capable of hybridizing thereto, wherein the 5 'end of the second amplification primer has a second recognition sequence for a second restriction endonuclease, and the endonuclease has two recognition sequences. (E) allowing the hybridized second amplification primer to nick one strand of the semi-modified recognition site of the strand; A deoxyribonucleoside triphosphate is extended from the 3 'end, i.e., to form a second double-stranded polynucleotide comprising a modified second polynucleotide extension product hybridized to the modified first polynucleotide extension product. A modified deoxyribonucleoside triphosphate, and a reaction mixture comprising a mixture of second polymerases capable of polymerizing deoxyribonucleoside triphosphate in the presence of the polynucleotide template and displacing the strand, An extension of a hybridized second amplification primer from its 3 'end, wherein the modified second polynucleotide extension product is separated from a single-stranded polynucleotide, and wherein the second recognition sequence and the first A copy of the target nucleic acid sequence located at the opposite end between the complement of the recognition sequence and a modified The complement of the first recognition sequence in the first polynucleotide extension product and the first recognition sequence in the second polynucleotide extension product is a double-stranded, semi-modified recognition site for the first recognition endonuclease. (F) nick one strand of the double-stranded semi-modified recognition site for the first recognition endonuclease, ie, the first restriction endonuclease, deoxyribonucleoside. A double-stranded, semi-modified, for the first recognition endonuclease in the presence of a triphosphate, a modified deoxyribonucleoside triphosphate, and the second synthase, to one strand of the recognition site. Nicking, whereby the nicked 3 ′ end is extended, thereby extending the modified first polynucleotide downstream thereof. Any portion of the long product can be replaced and defined at opposite ends by the first and second recognition sites, which can nick for the first and second restriction endonucleases, respectively. Forming a third double-stranded polynucleotide comprising a separated copy of the target nucleic acid sequence hybridized to a separated copy of the complement of the target nucleic acid sequence; (g) forming a separated copy of the target nucleic acid sequence or its complement. A third double-stranded polynucleotide in the presence of a mixture of deoxyribonucleoside triphosphates and modified deoxyribonucleoside triphosphates, an enzyme having DNA polymerase activity capable of amplifying, i.e., displacing the strand; By contacting the first restriction endonuclease with the second restriction endonuclease, the target nucleic acid sequence or its Amplifying an isolated copy of the complement, thereby nicking the first and second recognition sequences of the first and second recognition sites and extending the 3 'end of the nicked sites. A new target nucleic acid sequence or its complement is produced, and a downstream copy of the target nucleic acid sequence or its complement is replaced during the process; and (h) a detectable amount of the isolated copy of the target nucleic acid sequence is obtained. Continuing step (g) for a time sufficient to produce, comprising: amplifying a copy of the target nucleic acid sequence found in the single-stranded polynucleotide. 29. A method for detecting the presence of a target nucleic acid sequence found in a single-stranded polynucleotide, the method comprising: (a) preparing a reaction mixture, the reaction mixture comprising: (i) a single-stranded polynucleotide A sample containing the target nucleic acid sequence at a position other than the terminal end thereof or containing the target nucleic acid sequence; (ii) using a single-stranded polynucleotide as a template to extend the hybridized primer First polymerizing enzyme capable of polymerizing deoxyribonucleoside triphosphate, and deoxyribonucleoside triphosphate can be polymerized by extending a hybridized primer, and further, if the target nucleic acid is DNA, displacing the strand. A second polymerizing enzyme, wherein the first polymerizing enzyme can optionally be the same as the second polymerizing enzyme. And (iii) a mixture of deoxyribonucleoside triphosphates containing modified deoxyribonucleoside triphosphates; (iv) capable of hybridizing to a target nucleic acid sequence and forming a primer hybridized thereto. A first amplification primer comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, wherein the 3 ′ end of the first amplification primer is complementary to the 3 ′ end of the target nucleic acid sequence; The 5 'end of the first amplification primer can nick one strand of a double-stranded semi-modified recognition site for a first restriction endonuclease. A first amplification primer having a recognition sequence; and (v) a second amplification primer comprising an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, The 3 'end of the second amplification primer is complementary to, and can hybridize to, the 3' end of the complement of the target nucleic acid sequence, and the 5 'end of the second amplification primer is A restriction endonuclease having a second recognition sequence for a second restriction endonuclease, the endonuclease capable of nicking one strand of the double-stranded semi-modified recognition site that it recognizes; (B) applying the reaction mixture to a means for separating the double-stranded nucleic acids in the reaction mixture into single-stranded; (c) hybridization, primer extension, and The reaction mixture is allowed to stand for a time sufficient to perform nicking, thereby containing two separate copies of the target nucleic acid sequence and the complement of the target nucleic acid sequence, suitable for amplification. A polynucleotide extension product is produced, the double-stranded polynucleotide extension product comprising, at opposite ends, a nicked first restriction site and a second restriction site for each of the first and second restriction endonucleases. (D) an enzyme capable of amplifying an isolated copy of the target nucleic acid sequence or its complement, ie having DNA polymerase activity and displacing the strand, deoxyribonucleoside triphosphates and modified deoxyribose. By contacting the double-stranded polynucleotide extension product with the first restriction endonuclease and the second restriction endonuclease in the presence of a mixture of nucleoside triphosphates, an isolated copy of the target nucleic acid sequence or its complement is obtained. Amplifying, thereby, said first and second recognition of said first and second recognition sites The sequence is nicked and the 3 'ends of these nicked sites are extended to produce a new target nucleic acid sequence or its complement, and the downstream copy of the target nucleic acid sequence or its complement is processed by the process. (E) continuing step (d) for a time sufficient to produce a detectable amount of a separate copy of the target nucleic acid sequence; and (f) a detectable amount of the target nucleic acid sequence. Determining the presence of an isolated copy of the target nucleic acid sequence. The method for detecting the presence of a target nucleic acid sequence found in a single-stranded polynucleotide. 30. A method for determining the presence of a target nucleic acid sequence found in a single-stranded polynucleotide, comprising: (a) first amplifying at the 3 'end of the target nucleic acid sequence within the single-stranded polynucleotide; Hybridizing the primers, i.e., hybridizing the first amplification primer to the 3 'end of the target nucleic acid sequence within the single-stranded polynucleotide to form a hybridized first amplification primer, The first amplification primer includes an oligodeoxyribonucleotide having a 3 ′ end and a 5 ′ end, and the 3 ′ end of the first amplification primer is complementary to the 3 ′ end of the target nucleic acid sequence, The 5 'end of the first amplification primer has a first recognition sequence for a first restriction endonuclease, which endonuclease is capable of recognizing the double-stranded, semi-modified recognition site that it recognizes. (B) extending the hybridized first amplification primer from its 3 ′ end to complement and bind to the single-stranded polynucleotide; Using the target nucleic acid sequence as a template to form a first double-stranded polynucleotide having a modified first polynucleotide extension product, a deoxyribonucleoside triphosphate, a modified deoxyribonucleoside triphosphate, and Extending a hybridized first amplification primer from its 3 'end in the presence of a first polymerase capable of polymerizing deoxyribonucleoside triphosphate; (c) from the modified first polynucleotide extension product: Separating said single-stranded polynucleotide; (d) a second amplification primer at an internal site of said modified first polynucleotide extension product; Hybridizing the second amplification primer to an internal site of the modified first polynucleotide extension product to form a hybridized second amplification primer, wherein the second amplification primer comprises: The primer comprises an oligodeoxyribonucleotide with a 3 ′ end and a 5 ′ end, wherein the 3 ′ end of the second amplification primer is complementary to and 3 ′ end of the complement of the target nucleic acid sequence. The 5 'end of the second amplification primer has a second recognition sequence for a second restriction endonuclease, wherein the endonuclease recognizes a double-stranded (E) extending the hybridized second amplification primer from its 3 ′ end by nicking one strand of the semi-modified recognition site; That is, to form a second double-stranded polynucleotide comprising a modified second polynucleotide extension product hybridized to a modified first polynucleotide extension product, a deoxyribonucleoside triphosphate, a modified deoxyribonucleoside triphosphate An acid, and from its 3 ′ end in a reaction mixture comprising a mixture of second polymerases capable of polymerizing deoxyribonucleoside triphosphates in the presence of the polynucleotide template and displacing the strand. An extension of the hybridized second amplification primer, wherein the modified second polynucleotide extension product is separated from the single-stranded polynucleotide, and comprises the second recognition sequence and the complement of the first recognition sequence. A modified first polynucleotide comprising a copy of said target nucleic acid sequence located at opposing ends between The complement of the first recognition sequence in the otide extension product and the first recognition sequence in the second polynucleotide extension product forms a double-stranded, semi-modified recognition site for the first recognition endonuclease. (F) nicking one strand of the double-stranded, semi-modified, recognition site for the first recognition endonuclease, ie, the first restriction endonuclease, deoxyribonucleoside triphosphate. Nicking one half of the double-stranded, semi-modified recognition site for the first recognition endonuclease in the presence of a modified deoxyribonucleoside triphosphate, and the second synthase. In turn, this extends the nicked 3 ′ end, thereby reducing any of the modified first polynucleotide extension products downstream thereof. The target, defined at opposite ends by the first and second recognition sites, which can also be substituted and nickable for each of the first and second restriction endonucleases. Forming a third double-stranded polynucleotide comprising a separated copy of the target nucleic acid sequence hybridized to a separated copy of the complement of the nucleic acid sequence; (g) amplifying the separated copy of the target nucleic acid sequence or its complement; A third double-stranded polynucleotide, in the presence of a mixture of an enzyme having DNA polymerase activity capable of displacing the strand, deoxyribonucleoside triphosphate and modified deoxyribonucleoside triphosphate, Contacting the nuclease with the second restriction endonuclease to separate the target nucleic acid sequence or its complement The first and second recognition sequences of the first and second recognition sites are nicked, the 3 'ends of these nicked sites are extended, and the new target nucleic acid is amplified. The sequence or its complement is formed and the downstream copy of the target nucleic acid sequence or its complement is replaced during the process; (h) sufficient to produce a detectable amount of the separated copy of the target nucleic acid sequence Continuing step (g) over time; and (i) determining the presence of a detectable amount of a separated copy of the target nucleic acid sequence, comprising the steps of: Method for determining the existence of 31. 29. The method according to claim 28, wherein said amplification step (g) and said continuation step (h) proceed under substantially isothermal conditions. 32. 32. The method of claim 31, wherein said amplifying step (g) and said continuation step (h) proceed at about 37C to about 60C. 33. 33. The method according to claim 32, wherein said amplification step (g) and said continuation step (h) proceed at a temperature of about 60 <0> C.